人乳腺癌细胞系MCF-7原位移植血管生成超微结构观察及其相关因子表达

目的:探讨人乳腺癌细胞增殖与血管生成方式和时间的超微结构特点,明确人乳腺癌生长中VEGF、F8因子表达水平及其意义。方法:应用人乳腺癌细胞系MCF-7原位移植 SCID鼠,光镜、电镜动态观察肿瘤生长与血管生成的细胞形态。结合免疫组化检测VEGF、F8因子的表达水平。结果:人乳腺癌细胞系MCF-7移植后1、2天生存靠固有组织的血管滋养生长,肿瘤组织中未见新生血管生成。肿瘤生长第3天时,肉眼可见周围血管向移植的肿瘤表面延伸,电镜下观察在存活的癌细胞中可见血管母细胞出现。第6天时,在癌细胞增生活跃区血管母细胞演化为血管内皮细胞。此时癌细胞群与新生的肿瘤微血管紧密相连。微血管邻旁周边可见极少量的蛋白质网架。第8～10天,内皮细胞有较丰富的线粒体,粗面内质网增多,池内含中等量蛋白质,血管内皮的腔面小球增多,光镜下可见肿瘤周围的血管与肿瘤新生血管相通。VEGF、F8因子的表达与肿瘤增殖、侵袭、新生血管生成有关。结论:移植初期人乳腺癌细胞可分泌血管生成因子。肿瘤周围的血管母细胞被激活。快速增殖期癌细胞可诱导血管内皮细胞增生形成肿瘤新生血管。逐步演化的肿瘤新生血管与肿瘤边缘固有的血管相连通。     

RNA干扰MDM2下调人乳腺癌细胞中VEGF的合成并抑制肿瘤血管的生成

目的 探讨小干扰RNA（small interfering RNA, siRNA）抑制人乳腺癌细胞中鼠双微粒体2 （murine double minute 2, MDM2）的表达对癌细胞中血管内皮生长因子（vascular endothelial growth factor, VEGF）合成以及裸鼠移植瘤组织内血管生成的影响。方法 根据MDM2已知的cDNA序列设计并转录合成特异性siRNA,转染高表达MDM2的人乳腺癌MDA-MB-468细胞,低氧培养后应用蛋白质印迹法和ELISA法检测肿瘤细胞及其上清液中VEGF的水平。构建裸鼠乳腺癌移植瘤模型,观察MDM2沉默后肿瘤生长情况,蛋白质印迹法、免疫组织化学及ELISA方法检测荷瘤组织和裸鼠血清标本中的VEGF含量,并以CD34标记血管内皮细胞计数微血管密度（microvessel density, MVD）。结果 siRNA抑制MDM2表达后,MDA-MB-468细胞分泌的VEGF蛋白显著减少（P=0.006）。裸鼠移植瘤模型显示,封闭MDM2表达使荷瘤组织生长变慢（P=0.008）,且荷瘤小鼠血清VEGF水平明显减低（P=0.008）,荷瘤组织内MVD也明显降低（P=0.003）。结论 MDM2 siRNA能有效减低乳腺癌细胞中VEGF的合成,并抑制裸鼠移植瘤组织内新生血管的生成,为MDM2/VEGF途径抗肿瘤血管生成作用的研究及靶向药物开发提供了新的思路。     

人肺腺癌细胞LALU血管生成过程的形态学及超微结构观察

目的 观察人肺癌细胞血管生成过程的病理形态学、超微结构特点。方法 采用人肺腺癌细胞株LALU经SICO鼠皮下移植瘤模型,用光镜和电镜动态观察不同时期肿瘤血管生成状态。结果 光镜显示,人肺腺癌移植瘤第2－10天时可分为血管生成前期和血管形成期,第20天出现肺转移灶。电镜显示,人肺腺癌移植瘤第2天出现成血管细胞;第4－10天,不成熟血管内皮细胞逐渐形成血管腔并伴有较完整的新生基底板,内皮细胞趋向成熟发展;第20天肿瘤血管内皮细胞更成熟,部分区域新生毛细血管基底板发育不或缺陷。其全过程中癌细胞突起直接与成血管细胞、血管内皮细胞及血管壁相连。结论 病理形态学及电镜的特征形态学改变,提示肿瘤血管生成与转移有关,可为肺癌的血管导向治疗提供重要依据。     

贝伐单抗治疗恶性肿瘤相关不良反应的发生机制及处理方法

肿瘤生长和增殖过程中需要新生血管的形成,而血管内皮生长因子(VEGF)在其中起重要的作用.VEGF家族包括VEGF-A、VEGF-B、VEGF-C、VEGF-D等多个相关因子,而在肿瘤新生血管形成中最重要的是VEGF-A因子,它可促进血管内皮细胞生长、增殖,并与血管内皮细胞产生的生长因子受体相结合,激活下游信号转导通路,最终促进新生血管的生成.贝伐单抗(Bevacizumab)是与VEGF结合的重组人源化单克隆抗体,能与VEGF-A结合,阻止其与VEGF受体的相互作用,起到抗新生血管形成的作用,进而抑制肿瘤生长.     

重组人血管内皮抑素联合重组人P53腺病毒对裸鼠乳腺癌移植瘤的抑制

摘 要 目的: 探讨重组人血管内皮抑素（recombinant human endostatin,ES）联合重组人P53腺病毒（rAd/P53）对乳腺癌细胞MCF7裸鼠移植瘤的抑制作用。方法：建立裸鼠荷人乳腺癌模型，随机分为对照组、ES组、rAd/P53组和两药联合组,分别给予相应治疗；观察肿瘤生长，免疫组化法检测肿瘤组织微血管密度（microvessel density，MVD）和血管内皮生长因子（vascular endothelial growth factor, VEGF）的表达。结果：各治疗组均可明显抑制移植瘤生长， ES组、rAd/P53组和联合用药组的抑瘤率分别为51.67％、48.74％和75.54％，联合用药组抑瘤作用最为显著（P<0.05）。对照组、ES组、rAd/P53组与联合用药组的移植瘤组织MVD分别为31.17±2.48、20.33±4.84、22.33±3.88及12.50±2.74，VEGF 表达H评分分别为45.33±589、35.83±546、33.67±4.80及22.33±4.41，联合用药组的MVD和VEGF表达显著低于其余各组（P<0.01）。 结论：重组人ES联合rAd/P53治疗可显著抑制裸鼠乳腺癌移植瘤的生长及微血管生成。     

BCYRN1对乳腺癌细胞MCF7和小鼠移植瘤增殖和转移的影响

目的 研究RNA干扰lncRNA BCYRN1对乳腺癌细胞MCF7和小鼠移植瘤的增殖和转移的影响。 方法 BCYRN1 siRNA和siRNA scramble（对照组）分别转染乳腺癌细胞MCF7。CCK-8检测细胞增殖;蛋白印迹法检测细胞中蛋白的表达;划痕实验分析细胞运动能力;免疫组织化学法检测肿瘤组织中蛋白的表达。 结果 与对照组相比,细胞增殖倍数明显降低（P=0.02）;且细胞增殖核抗原-67（antigen identified by monoclonal antibody, Ki-67）和增殖细胞核抗原（proliferating cell nuclearantigen, PCNA）表达显著减弱（P=0.03）。BCYRN1 siRNA处理后,乳腺癌MCF7细胞运动能力减弱,血管内皮细胞生长因子（vascular endothelial growth factor, VEGF）和基质金属蛋白酶9（matrixmetalloprotein 9, MMP-9）表达受到抑制（P=0.04）。体内实验表明,BCYRN1 siRNA组小鼠肿瘤的体积明显小于对照组（P=0.03）,且BCYRN1 siRNA可以抑制MCF7小鼠移植瘤肿瘤组织Ki-67和MMP-9的表达。 结论 BCYRN1 siRNA可以抑制乳腺癌细胞MCF7和小鼠的增殖和转移     

血管内皮生长因子C在人乳腺癌细胞株MCF-7及MCF-7/Adr中的表达

背景与目的：血管内皮生长因子C（vascular endothelial growth factor C,VEGF-C)是近期发现的血管内皮生长因子（vascular endothelial growth factor,VEGF)家族的新成员。研究发现VEGF－C可特异性作用于淋巴管内皮细胞,并在多种人类肿瘤中表达。为探索VEGF－C在肿瘤发生,发展中的作用,我们分别检测了VEGF－C mRNA蛋白在人乳腺癌细胞株MCF－7及其耐药株MCF－7／Adr中的表达。方法;根据VEGF－C基因序列,设计合成地高辛标记的特异性寡核 探针,运用原位杂交方法检测培养的细胞株MCF－7和MCF－7／Adrk VEGF-C mRNA的表达,并运用免疫组织化学方法检测两种细胞中VEGF－C蛋白的表达,实验中用缓冲液分别代替杂交液和一抗作阴性对照。结果：原位杂交法检测到MCF－7和MCF－7／Adr细胞的胞浆中有阳性蓝色颗粒,免疫组化检测发现两种细胞的胞浆中均有阳性棕黄色颗粒,而阴性对照细胞的胞浆中则均无阳性颗粒。结论：人乳腺癌细胞株MCF－7及其耐药株MCF－7／Adr细胞能够转录VEGF-C mRNA,并在其细胞浆中翻译合成相应的蛋白。     

双调蛋白反义核酸表达对裸鼠乳腺癌血管生成的抑制作用

目的 研究双调蛋白（AR）反义RNA表达对乳腺癌血管生成的抑制作用。方法 乳腺癌NS2T2A1细胞经AR反义cDNA质粒转染后,经筛选获得表达AR反义RNA的AR-AS1及AR-AS3克隆,转染空载体获得NS2T2A1 V对照细胞,接种裸鼠皮下形成肿瘤。研究条件培养液对人微血管内皮细胞（HMEC）增殖的影响,以EL1SA法测定细胞血管内皮生长因子（VEGF）分泌量。以定量RT-PCR分析肿瘤组织的VEGF表达水平。以免疫组化法标记CD31研究肿瘤内血管数量。结果 HMEC在AR-AS1和AR-AS3细胞条件培养液中的增殖比例明显降低。AR-AS1和AR-AS3细胞VEGF分泌量亦降低。AR-AS1和AR-AS3肿瘤的血管数量仅有对照组的50％左右,且VEGF表达显著降低。结论 AR反义RNA表达可有效抑制乳腺癌的血管生成,应作为新治疗靶点进一步研究．     

抗VEGF发卡状核酶抑制肝癌VEGF表达和移植瘤生长

目的:采用抗人血管内皮生长因子(vascular endothelial growth factor,VEGF)发卡状核酶基因转染肝癌细胞,观察核酶对VEGF表达及肿瘤生长的影响。方法:采用脂质体介导的方法,将人工合成的抗VEGF发卡状核酶基因转染肝癌细胞SMMC-7721,同时制备空载体和细胞对照,经G418筛选获得阳性克隆,基因组水平鉴定核酶在细胞中的表达,半定量RT-PCR法和免疫组化法观察核酶对SMMC-7721细胞VEGF表达的影响。接种各组细胞于裸鼠皮下,观察瘤体体积大小和质量,免疫组化法观测各组肿瘤微血管变化和VEGF的表达。结果:核酶基因成功导入到肿瘤细胞中,转基因细胞的增殖速率明显下降(P<0.01),转染核酶组细胞VEGF水平明显下降(P<0.01)。移植瘤成瘤率和生长速度减慢(P<0.01),肿瘤组织VEGF表达和血管形成减少(P<0.01)。结论:抗人VEGF发卡状核酶基因在体内、体外均可抑制肝癌VEGF表达,通过减少血管形成抑制肿瘤生长,为进一步开展肝癌血管靶向基因治疗提供实验依据。     

乳腺癌血管内皮生长因子与微血管密度的免疫组织化学研究

 血管内皮生长因子（VEGF）是特异作用于血管内皮细胞、上调血管生成的重要因子，能刺激血管内皮细胞增殖、迁徙和诱导血管生成，而血管新生是实体瘤生长、浸润、转移与复发的重要前提。本实验采用免疫组织化学SP法对48例乳腺癌组织中VEGF表达、MVD与乳腺癌生物学行为间的关系进行探讨。     

Screening of antiangiogenic compound

Cancer cells promote angiogenesis early in tumorigenesis because cancer cells require oxygen and nutrients for their survival they need to be supported by blood vessels. The angiogenic switch is driven by several proangiogenic factors, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF) and others. These proangiogenic factors are released from not only cancer cells but also various stromal cells by various stimulations, mutations or inflammation. From our experimental results using human lung cancer cell lines, survival factors in each cancer cell line are different. This makes cancer therapy difficult. Various angiogenesis inhibitors have been developed to block tumor angiogenesis. However we need to think how and when to use angiogenesis inhibitors together with conventional chemotherapeutic agents.     

Androgen withdrawal inhibits tumor growth and is associated with decrease in angiogenesis and VEGF expression in androgen-independent CWR22Rv1 human prostate cancer model

Recent evidence suggests that androgens stimulate growth of human prostate cancer partly by regulating expression of growth factors such as vascular endothelial growth factor (VEGF) in vitro and in vivo. In this study, we used CWR22Rv1, a novel androgen-responsive but androgen-independent human prostate cancer model, to evaluate the effect of androgen withdrawal on tumor growth, expression of VEGF and the cell proliferation marker Ki-67, and angiogenesis. A time-release testosterone pellet was implanted three days before inoculation of CWR22Rv1 cells in the mice. The tumor volumes were measured every three days. Serum PSA was measured on days 1, 12, 20, 27 and 34 post inoculation. Castration was performed on the 20th day post inoculation. Immunohistochemical assays were used to evaluate cell proliferation and microvessel density. Enzyme-linked immunosorbent assay (ELISA) was used to quantify VEGF expression. The average tumor volumes in the castration group on the 27th and 34th days were 122 and 168 mm3, respectively, compared to 156 and 210 mm3 in the non-castration group (p<0.01). Serum PSA level in the castration group decreased to about 41% of the level of the non-castration group (p<0.01). The VEGF protein levels in the tumors of castrated and non-castrated mice on day 34 were 0.62 pg and 1.36 pg/100 microg total protein, respectively (p<0.001). The mean percentage of Ki-67-positive tumor cells in the castrated and non-castrated groups were 1.8% and 2.8%, respectively (p=0.015). The mean microvessel densities in the castrated and non-castrated groups were 15 and 22 vessels/field, respectively (p<0.01) We conclude androgen withdrawal reduced both VEGF and microvessel density, and this was associated with decreased cellular proliferation in androgen-independent CWR22Rv1 human prostate cancer tumor in vivo.     

The histone deacetylase inhibitor NVP-LAQ824 inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584

Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. Vascular endothelial growth factor (VEGF) and VEGF receptors represent critical molecular targets for antiangiogenesis therapy. In this study, we investigated the biological effect of the histone deacetylase inhibitor NVP-LAQ824 in combination with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on tumor growth and angiogenesis. We report that treatment with NVP-LAQ824 affected tumor and endothelial cells and was associated with increased histone acetylation, p21 up-regulation, and growth inhibition. In addition, NVP-LAQ824 treatment inhibited the expression of angiogenesis-related genes such as angiopoietin-2, Tie-2, and survivin in endothelial cells and down-regulated hypoxia-inducible factor 1-alpha and VEGF expression in tumor cells. Combination treatment with NVP-LAQ824 and PTK787/ZK222584 was more effective than single agents in inhibiting in vitro and in vivo VEGF-induced angiogenesis. Endothelial cell proliferation, tube formation, and invasion into the Matrigel plugs were reduced. In mouse models with established subcutaneous prostate (PC3) and orthotopic breast tumors (MDA-MB321), this combination treatment induced 80 to 85% inhibition of tumor growth without overt toxicity. These results suggest that the combination of histone deacetylase inhibitors and VEGF receptor inhibitors may target multiple pathways in tumor progression and angiogenesis and represents a novel therapeutic approach in cancer treatment.     

Tumor suppressor XAF1 induces apoptosis,inhibits angiogenesis and inhibits tumor growth in hepatocellular carcinoma

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1), a XIAP-binding protein, is a tumor suppressor gene. XAF1 was silent or expressed lowly in most human malignant tumors. However, the role of XAF1 in hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the effect of XAF1 on tumor growth and angiogenesis in hepatocellular cancer cells. Our results showed that XAF1 expression was lower in HCC cell lines SMMC-7721, Hep G2 and BEL-7404 and liver cancer tissues than that in paired non-cancer liver tissues. Adenovirus-mediated XAF1 expression (Ad5/F35-XAF1) significantly inhibited cell proliferation and induced apoptosis in HCC cells in dose- and time- dependent manners. Infection of Ad5/F35-XAF1 induced cleavage of caspase -3, -8, -9 and PARP in HCC cells. Furthermore, Ad5/F35-XAF1 treatment significantly suppressed tumor growth in a xenograft model of liver cancer cells. Western Blot and immunohistochemistry staining showed that Ad5/F35-XAF1 treatment suppressed expression of vascular endothelial growth factor (VEGF), which is associated with tumor angiogenesis, in cancer cells and xenograft tumor tissues. Moreover, Ad5/F35-XAF1 treatment prolonged the survival of tumor-bearing mice. Our results demonstrate that XAF1 inhibits tumor growth by inducing apoptosis and inhibiting tumor angiogenesis. XAF1 may be a promising target for liver cancer treatment.     

Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ

Antiangiogenic therapy shows promise as a strategy for cancer treatment. We constructed an adenovirus (AdVEGF-ExR) expressing the entire extracellular domain of the human vascular endothelial growth factor (VEGF) receptor (flt-1) fused to the Fc portion of human IgG. The soluble receptor secreted from AdVEGF-ExR-infected cells bound to VEGF and inhibited VEGF-induced DNA synthesis in endothelial cells. When human lung cancer cell line H157, which produces not only VEGF but also fibroblast growth factor 2 and interleukin 8 at substantial levels, was infected with AdVEGF-ExR, cell growth in vitro was not affected. However, when H157 cells infected with AdVEGF-ExR were injected s.c. into nude mice, tumor formation stopped on the 10th day after reaching a certain size (about 100 mm3), and tumor size declined gradually thereafter. When AdVEGF-ExR was injected into skeletal muscle and uninfected H157 cells were injected s.c., the soluble receptor was detectable in the circulating blood for 3 weeks, tumor growth ceased after 10 days, and tumor size declined thereafter. Histological examination revealed that intratumor angiogenesis was markedly suppressed, and apoptosis was enhanced. Using the same experimental protocol, a significant suppression of tumor growth was also seen in four of five other lung cancer cell lines, some of which secreted VEGF at nominal levels, at least under normoxic conditions in vitro. Our results demonstrate that adenovirus-mediated expression of a soluble VEGF receptor in a remote organ could inhibit tumor angiogenesis and enhance apoptosis and thereby suppress tumor growth in vivo. Adenovirus-mediated overexpression of a soluble VEGF receptor in a remote organ may have the potential to be a feasible and effective strategy for cancer treatment.     

Effect of VEGF and VEGF Trap on vascular endothelial cell signaling in tumors

Vascular endothelial growth factor (VEGF) A is a major promoter of tumor angiogenesis and a prime target of antiangiogenic cancer therapy. To examine whether endothelial cell signaling might provide histological biomarkers of angiogenesis and VEGF activity in vivo, normal mouse organs and multiple tumor models were studied immunohistochemically for endothelial expression of activated ERK, STAT3 and AKT. Phospho(p)-ERK and p-STAT3 expression was negligible in the endothelia of normal organs but was significantly elevated in tumor endothelium. p-AKT was present at significant and comparable levels in both tumor and normal endothelia. In K1735 tumors induced to express more VEGF, endothelial p-ERK, p-STAT3 and p-AKT increased accompanied by signs of accelerated angiogenesis. Treatment of K1735 and Colo-205 tumors with the VEGF inhibitor, VEGF Trap (aflibercept), decreased tumor endothelial p-ERK, p-STAT3 and p-AKT expression accompanied by signs of antiangiogenic effect. These results show that endothelial p-ERK and p-STAT3 (but not p-AKT) distinguish tumor from normal vessels and that the presence of these two signaling intermediates may be useful indicators of tumor angiogenic activity and angiogenesis inhibition by VEGF antagonists.Key words: VEGF, VEGF Trap, endothelial cells, signal transduction, angiogenesis, biomarker, p-ERK, p-STAT3, p-AKT     

Fibulins 3 and 5 antagonize tumor angiogenesis in vivo

Lethal tumor growth and progression cannot occur without angiogenesis, which facilitates cancer cell proliferation, survival, and dissemination. Fibulins (FBLN) 5 and 3 are widely expressed extracellular matrix proteins that regulate cell proliferation in a context-specific manner. Reduced FBLN-5 expression has been associated with cancer formation and progression in humans, whereas its constitutive expression antagonizes endothelial cell angiogenic sprouting in vitro. Thus, FBLN-5 may suppress tumorigenesis by preventing tumor angiogenesis. FBLN-3 is homologous to FBLN-5 and expressed in endothelial cells, yet its role in tumorigenesis and angiogenesis is unknown. We find FBLN-3 expression to be altered in some human tumors and that its constitutive expression in endothelial cells inhibited their proliferation, invasion, and angiogenic sprouting, as well as their response to vascular endothelial growth factor as measured by p38 mitogen-activated protein kinase activation. In endothelial cells, both FBLNs (a) reduced angiogenic sprouting stimulated by basic fibroblast growth factor (bFGF); (b) inhibited matrix metalloproteinase expression and activity; and (c) stimulated tissue inhibitor of metalloproteinase expression. More importantly, both FBLNs prevented angiogenesis and vessel infiltration into bFGF-supplemented Matrigel plugs implanted in genetically normal mice, as well as decreased the growth and blood vessel density in tumors produced by MCA102 fibrosarcoma cells implanted s.c. into syngeneic mice. Our findings establish FBLN-3 and FBLN-5 as novel angiostatic agents capable of reducing tumor angiogenesis and, consequently, tumor growth in vivo and suggest that these angiostatic activities may one day be exploited to combat tumor angiogenesis and metastasis in cancer patients.