Effects of tumor cell-derived interleukin 1 alpha on invasiveness of metastatic clones of murine RCT sarcoma through endothelial cells.

Interleukin 1 alpha (IL-1alpha) production and invasiveness through mouse lung endothelial cells (MLE) were investigated in high-metastatic RCT+ and low-metastatic RCT- clones established from poorly differentiated murine sarcoma. Apparently, a higher level of IL-1alpha was derived from RCT+ cells than from RCT- cells. In an invasion assay, the number of cells which penetrated the MLE monolayer in RCT+ was significantly greater than that in RCT-. The invasiveness of RCT+ and RCT- cells was stimulated by additional recombinant mouse IL-1alpha (rIL-1alpha) in a dose-dependent manner. Anti-mouse IL-1alpha monoclonal antibody (anti-IL-1alpha mAb) significantly inhibited the invasiveness of RCT+ and RCT- cells through the MLE monolayer. However, in RCT+ cells these effects were higher than in RCT- cells. In an attachment assay, the ability of RCT+ cells to attach to the MLE monolayer was significantly higher than that of RCT- cells. The attachment ability of RCT+ and RCT- cells to the MLE monolayer was significantly increased by the pretreatment with rIL-1alpha in a dose-dependent manner. In a retraction assay, conditioned medium of RCT+ stimulated the retraction of the MLE monolayer more markedly in comparison with conditioned medium of RCT-. The retraction of the MLE monolayer was stimulated by additional rIL-1alpha in a dose-dependent manner. The increased retraction of the MLE monolayer was closely associated with the enhancement in tumor cell invasiveness. These findings suggest that IL-1alpha derived from RCT+ and RCT- cells might contribute to the enhancement of tumor cell invasion by stimulating the attachment to the MLE monolayer and retraction of the MLE monolayer.     

Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. K562 human leukemia cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. In the present study, three 19 bp reverse repeated motifs targeting exons 5 and 7 boundary of VEGF165 gene sequence with 9 bp spacer were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into K562 cells, respectively, through lipofectamine reagent. A vector-based small interfering RNA(SiRNA) inhibited VEGF165 mRNA expression by 72% and protein production by 67% in K562 cells. Human microvascular endothelial cell migration induced by conditioned medium from VEGFsi-transfected K562 cells was significantly less than that induced by conditioned medium from K562 cells and control vector-transfected K562 cells. Furthermore, the VEGF shRNA dramatically suppressed tumor angiogenesis and tumor growth in a K562 s.c. xenograft model. Vessel density as assessed by vWF immunohistochemical analysis was also decreased. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF-specific treatment in cancer.     

Characterization and purification of angiogenic factor derived from highly liver metastatic colon cancer cells

We investigated the effects of serum-free conditioned medium (SFCM) of colon cancer cells on human umbilical vein endothelial cells (HUVECs). SFCM of highly liver metastatic cell line (LM-HS) strongly enhanced proliferation, migration and tube formation of HUVECs. Moreover, it was suggested that a main angiogenic factor derived from LM-H5 may be VEGF based on HPLC, SDS-PAGE analysis and neutralizing experiment using anti-growth factor antibodies. These findings suggest that angiogenesis is related to the processes of metastasis of colon cancer, and that VEGF synthesized by colon cancer cells acts as a key factor inducing angiogenesis.     

Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.     

Continuous treatment of bestatin induces anti-angiogenic property in endothelial cells

CD13/aminopeptidase-N (CD13/APN) is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. A previous study demonstrated that angiogenesis is suppressed under the presence of high concentrations of aminopeptidase antagonists. However, the mechanisms underlying the inhibition of morphogenesis by aminopeptidase antagonists have not been elucidated. In this study, we have for the first time examined the effects of continuous treatment of therapeutic dose of aminopeptidase antagonists on vascular endothelial capillary-like tube formation. In the antagonists tested, only bestatin significantly interfered in the capillary tube formation of primary endothelial cells (EC) after treatment for 72 h. Aminopeptidase analysis revealed that inhibitory activity of bestatin was not specific for CD13/APN, and the other inhibitors lacking anti-angiogenic properties also inhibit cell-surface aminopeptidase activity as well or more potently than bestatin, suggesting that the angiogenesis-inhibitory effect of bestatin was not due to inhibition of CD13/APN activity at this concentration. To elucidate the influence of continuous treatment of bestatin on endothelial cells, we performed microarray analysis and revealed that 72-h treatment of a pharmacokinetic dose of bestatin modulated the several angiogenesis-related genes including vascular endothelial growth factor (VEGF). Northern blot analysis indicated that modulation of the VEGF gene became obvious after 48 h of treatment. Furthermore, knockdown of the VEGF gene by siRNA remarkably suppressed capillary tube formation and required a higher concentration of exogenous VEGF to reverse the capillary formation ability. These data suggested that bestatin decreases a reactivity of EC to angiogenesis stimuli, and it can be achieved by the regulation of angiogenesis-related gene expression.     

靶向肿瘤血管内皮细胞抑制人肝癌移植瘤生长的功能性抗体的研制

背景与目的:肿瘤的生长依赖于新生血管形成,阻断其血管形成可有效治疗肿瘤.本研究旨在研制抗人肝癌血管内皮细胞的功能性单克隆抗体,确定其抑制人肝癌移植瘤生长的作用.方法:用从新鲜人肝癌组织中分离、鉴定、培养的人肝癌血管内皮细胞免疫10只BALB/c小鼠.免疫小鼠脾细胞与SP2/0细胞融合后采用甲基纤维素选择培养.使用活细胞免疫荧光、细胞增殖、内皮成管实验及"人源化血管"动物模型体内抑瘤实验筛选和鉴定有治疗潜力的功能性抗体.结果:细胞融合产生1 442个单个集落的杂交瘤株,获得119株阳性克隆,其中53株是具有抑制人肝癌血管内皮细胞增殖、成管的功能性抗体,2株被证实能抑制人肝癌移植瘤在BALB/c裸鼠体内的生长,抑瘤率为66.7%～76.5%.结论:建立了高通量制备、筛选、鉴定内皮细胞功能性单抗技术,获得了2株具有靶向血管内皮细胞抑制人肝癌移植瘤生长的功能性单抗.     

Anti‐CD73 therapy impairs tumor angiogenesis

CD73 is an ecto‐nucleotidase overexpressed in various types of tumors that catabolizes the generation of extracellular adenosine, a potent immunosuppressor. We and others have shown that targeted blockade of CD73 can rescue anti‐tumor T cells from the immunosuppressive effects of extracellular adenosine. Another important function of extracellular adenosine is to regulate adaptive responses to hypoxia. However, the importance of CD73 for tumor angiogenesis and the effect of anti‐CD73 therapy on tumor angiogenesis remain unknown. In this study, we demonstrated that CD73 expression on tumor cells and host cells contribute to tumor angiogenesis. Our data revealed that tumor‐derived CD73 enhances the production of vascular endothelial growth factor (VEGF) by tumor cells that host‐derived CD73 is required for in vivo angiogenic responses and that endothelial cells require CD73 expression for tube formation and migration. Notably, the pro‐angiogeneic effects of CD73 relied on both enzymatic and non‐enzymatic functions. Using a mouse model of breast cancer, we demonstrated that targeted blockade of CD73 with a monoclonal antibody significantly decreased tumor VEGF levels and suppressed tumor angiogenesis in vivo. Taken together, our study strongly suggests that targeted blockade of CD73 can significantly block tumor angiogenesis, and further supports its clinical development for cancer treatment.     

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.     

Vascular endothelial growth factor overexpression by soft tissue sarcoma cells: implications for tumor growth, metastasis, and chemoresistance

To better elucidate the role of vascular endothelial growth factor (VEGF)(165) in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF(165) to study the effect of VEGF(165) on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF(165)-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases.     

Prostate cancer-derived CCN3 induces M2 macrophage infiltration and contributes to angiogenesis in prostate cancer microenvironment

Tumor-associated macrophages (TAMs) are M2-polarized macrophages that infiltrate the tumor microenvironment and promote tumorigenesis. However, the mechanisms by which TAMs modulate prostate cancer (PCa) growth are poorly understood. Here, we found that expression of Nephroblastoma Overexpressed (NOV/CCN3) is upregulated in PCa cells and correlated with M2 macrophage infiltration. RAW264.7 macrophage migration was induced by conditioned media (CM) from various PCa cells in proportion to the cellular level of CCN3 expression and was inhibited by an anti-CCN3 neutralizing antibody. CCN3 and PCaCM treatment skewed RAW264.7 cell differentiation from an M1 phenotype to an M2 phenotype. PCa-derived CCN3 induced focal adhesion kinase (FAK)/Akt/NF-κB signaling in RAW264.7 cells, which resulted in VEGF expression and subsequently increased tube formation in endothelial progenitor cells. Finally, PCa-secreted CCN3 stimulated RAW264.7 cells and promoted angiogenesis in the chick chorioallantoic membrane assay (CAM), and increased tumor growth and tumor-associated angiogenesis in a PCa xenograft mouse model. Our results indicate that PCa-secreted CCN3 can recruit macrophages and skew their differentiation to an M2 phenotype. In turn, CCN3-stimulated macrophages contribute to VEGF-dependent angiogenesis. This study reveals a novel mechanism by which TAMs enhance PCa angiogenesis and identifies a potential therapeutic target for PCa.     

Anti-angiogenic activity of inositol hexaphosphate (IP6)

A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP(6)) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP(6) reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP(6) on both. IP(6) inhibited the proliferation and induced the differentiation of endothelial cells in vitro; the growth of bovine aortic endothelial cells (BAECs) evaluated by MTT proliferation assay was inhibited in a dose-dependent manner (IC(50) = 0.74 mM). The combination of IP(6) and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP(6) inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP(6) significantly reduced basic fibroblast growth factor (bFGF)-induced vessel formation (P < 0.01) in vivo in Matrigel plug assay. Exposure of HepG2, a human hepatoma cell line, to IP(6) for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RT-PCR. IP(6) treatment of HepG2 cells for 24 h also significantly reduced the VEGF protein levels in conditioned medium, in a concentration-dependent manner (P = 0.012). Thus, IP(6) has an inhibitory effect on induced angiogenesis.     

腹膜间皮细胞对卵巢癌细胞血管生成因子表达及分泌的影响

目的探讨腹膜间皮细胞（HPMC）对卵巢癌细胞血管生成因子表达及分泌的影响。方法用ELISA法检测HPMC条件培养液中肿瘤坏死因子α（TNF—α）及白介素-1β（IL-1β）的水平。用培养小室Millicell将卵巢癌细胞SKOV3与HPMC在不同培养条件下进行共培养。用RT—PCR方法检测SKOV3血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）的基因表达,ELISA法检测SKOV3条件培养液中VEGF和bFGF的蛋白水平。结果HPMC条件培养液中可检测到TNF—α和IL-1β。SKOV3与HPMC共培养后,其VEGF和bFGFmRNA表达增强,条件培养液中VEGF和bFGF蛋白水平升高,与SKOV3单独培养相比,差异均有统计学意义（P〈0．01）。加入TNF—α或IL-1β的中和抗体共培养,可明显抑制SKOV3VEGF及bFGFmRNA表达及蛋白分泌（P〈0．01）,共培养体系中同时加入TNF—α中和抗体及IL-1β中和抗体时,抑制作用增强（P〈0．05）。结论HPMC分泌TNF—α和IL-1β,刺激卵巢癌细胞表达及分泌更高的VEGF和bFGF,参与卵巢癌的血管生成及腹膜转移。     

CXCL12–CXCR7 axis is important for tumor endothelial cell angiogenic property

We reported that tumor endothelial cells (TECs) differ from normal endothelial cells (NECs) in many aspects, such as gene expression profiles. Although CXCR7 is reportedly highly expressed in blood vessels of several tumors, its function in TECs is still unknown. To investigate this role, we isolated TECs from mouse tumor A375SM xenografts, and compared them with NECs from normal mouse dermis. After confirming CXCR7 upregulation in TECs, we analyzed its function using CXCR7 siRNA and CXCR7 inhibitor; CCX771. CXCR7 siRNA and CCX771 inhibited migration, tube formation and resistance to serum starvation in TECs but not in NECs. ERK1/2 phosphorylation was inhibited by CXCR7 knockdown in TECs. These results suggest that CXCR7 promotes angiogenesis in TECs via ERK1/2 phosphorylation. Using ELISA, we also detected CXCL12, a ligand of CXCR7, in conditioned medium from TECs, but not from NECs. CXCL12 neutralizing antibody significantly inhibited TEC random motility. VEGF stimulation upregulated CXCR7 expression in NECs, implying that VEGF mediates CXCR7 expression in endothelial cells. A CXCR7 inhibitor, CCX771 also inhibited tumor growth, lung metastasis and tumor angiogenesis in vivo. Taken together, the CXCL12–CXCR7 autocrine loop affects TEC proangiogenic properties, and could be the basis for an antiangiogenic therapy that specifically targets tumor blood vessels rather than normal vessels.     

20（S）-人参皂苷Rg3对血管内皮细胞增殖和迁移的抑制作用

 目的 探讨20（S）-人参皂苷Rg3（SPG-Rg3）抗肿瘤诱导新生血管生成的作用机制。方法 采用MTT法、PCNA免疫荧光染色和Boyden小室迁移实验,观察SPG-Rg3对B16黑色素瘤细胞条件培养液（Conditioned Medium of B16 Melanoma Cells, BMCM）诱导的人脐静脉内皮细胞增殖和迁移的影响;并通过免疫细胞化学染色法观察了SPG-Rg3对B16黑色素瘤细胞血管内皮细胞生长因子（Vascular Endothelial Growth Factor, VEGF）及金属基质蛋白酶-9（Matrix Metalloproteinase-9, MMP-9）表达的影响。结果SPG-Rg3组BMCM的促内皮细胞增殖和迁移作用均明显弱于对照组,且SPG-Rg3浓度为5μg/ml时能够降低B16黑色素瘤细胞VEGF及MMP-9的表达。结论SPG-Rg3通过减少肿瘤细胞分泌细胞因子VEGF和MMP-9,抑制肿瘤细胞对血管内皮细胞增殖和迁移的促进作用,从而间接发挥其抗肿瘤新生血管生成的作用。     

Detection of prostate-specific membrane antigen on HUVECs in response to breast tumor-conditioned medium

Prostate-specific membrane antigen (PSMA), a well-known biomarker of prostate cancer, has also been found to be highly expressed in the neovasculature of multiple non-prostatic solid tumors. As a consequence, it has the potential to become a biomarker for tumor-associated vasculature. Herein, we describe an in vitro model for assessing PSMA expression associated with tube formation by primary human umbilical vein endothelial cells (HUVECs) cultured in Matrigel and induced by tumor-conditioned medium (TCM) derived from human breast cancer cells (MDA-MB-231). In contrast to vascular endothelial growth factor (VEGF)-containing endothelial cell medium, TCM induced higher expression of PSMA in HUVECs. The vessel-like tubes were detected by imaging with fluorescent PSMA inhibitors. Consequently, this in vitro model is expected to enable subsequent studies aimed at determining the role of PSMA in angiogenesis and factors that induce it.