Hypoxia-induced metastasis of human melanoma cells: involvement of vascular endothelial growth factor-mediated angiogenesis.

Tumour cells exposed to hypoxia have been shown to up-regulate the expression of vascular endothelial growth factor (VEGF). The purpose of the present work was to investigate whether hypoxia-induced VEGF up-regulation can result in increased metastatic efficiency of human melanoma cells. Two melanoma lines, one showing high (A-07) and the other showing low (D-12) VEGF secretion under aerobic conditions, were included in the study. Cell cultures were exposed to hypoxia (oxygen concentrations < 10 ppm) in vitro and metastatic efficiency, i.e. lung colonization efficiency, as well as transplantability and angiogenic potential were assessed in BALB/c-nu/nu mice Both cell lines showed significantly increased VEGF secretion under hypoxic conditions as measured by enzyme-linked immunosorbent assay The D-12 cells showed increased metastatic efficiency, transplantability and angiogenic potential following exposure to hypoxia. The metastatic efficiency increased with the duration of the hypoxia treatment and decreased with the time after reoxygenation. The A-07 cells on the other hand showed unchanged metastatic efficiency, transplantability and angiogenic potential following exposure to hypoxia. Both cell lines showed significantly decreased metastatic efficiency and angiogenic potential in mice treated with neutralizing antibody against VEGF. These results suggest that (a) VEGF is a limiting factor for the rate of angiogenesis in low but not in high VEGF-expressing melanomas under normoxic conditions and (b) transient hypoxia might promote the development of metastases in low VEGF-expressing melanomas by upregulating the expression of VEGF and hence enhancing the angiogenic potential of the tumour cells.     

Anti‐VEGF antibody therapy induces tumor hypoxia and stanniocalcin 2 expression and potentiates growth of human colon cancer xenografts

Tumor angiogenesis plays a critical role in colorectal cancer progression. Recent randomized clinical trials have revealed the additive effect of bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF)‐A, to conventional chemotherapy in the improved survival of patients with metastatic colorectal cancer. However, a number of preclinical reports indicate the development of resistance to anti‐angiogenic therapy. In this study, we addressed the effects of anti‐VEGF antibodies on the growth and malignant behavior of colorectal cancer cells. TK‐4, a solid tumor strain derived from a colon cancer patient, was subcutaneously or orthotopically implanted into nude mice. Short‐term administration of anti‐VEGF antibodies inhibited the growth of cecal tumors at day 14 by suppressing mitosis, but prolonged treatment resulted in the recovery of cellular proliferation and suppression of apoptosis at day 35. Intratumoral hypoxia induced by anti‐VEGF antibody treatment resulted in activation of hypoxia inducible factor‐1α protein and an increased number of aldehyde dehydrogenase 1‐positive tumor cells. In microarray analysis, stanniocalcin 2 (STC2) was the most highly upregulated gene in anti‐VEGF antibody‐treated tumors. In vitro analyses showed that the growth and migration of SW480 colon cancer cells under hypoxic conditions were significantly inhibited by knockdown of STC2. In vivo serial transplantation of TK‐4 revealed that long‐term administration of anti‐VEGF antibodies increased the tumorigenicity of colon cancers and accelerated tumor growth when transplanted into secondary recipient mice. Our data provide a potential molecular explanation for the limited clinical effectiveness of anti‐VEGF antibodies.     

Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

The aim of this study was to investigate whether tumour hypoxia and/or vascular hot spots promote the development of metastatic disease. The D-12 human melanoma xenograft line was used as a tumour model. Hypoxia and vascular hot spots were detected by immunohistochemistry using pimonidazole as a hypoxia marker and anti-CD31 antibody to visualize endothelial cells. Vascular hot spots were found to be induced in hypoxic foci, owing to hypoxia-induced up-regulation of angiogenesis stimulatory factors. This effect was mediated by interleukin 8 and possibly also by vascular endothelial growth factor. Interleukin 8 positive foci showed a high degree of co-localization with hypoxic foci, as revealed by immunohistochemistry. The incidence of spontaneous pulmonary metastases was associated with the density of hypoxic foci, the density of interleukin 8 positive foci and the density of vascular hot spots in the primary tumour. Treatment with neutralizing antibody against interleukin 8 and/or vascular endothelial growth factor resulted in hypoxia-induced necrosis rather than hypoxia-induced vascular hot spots and inhibited metastasis. Our study suggests a cause-effect relationship between hypoxia and metastasis in cancer and hence an elevated probability of metastatic disease in patients having primary tumours characterized by high densities of hypoxic foci and vascular hot spots.     

The constitutive level of vascular endothelial growth factor (VEGF) is more important than hypoxia-induced VEGF up-regulation in the angiogenesis of human melanoma xenografts

Angiogenesis of tumours might develop as a result of environmental conditions, such as hypoxia, and/or as a result of genetic alterations specific for tumour cells. The relative contributions of these mechanisms were investigated by comparing the in vivo expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to the hypoxic fraction, the angiogenic potential and the vascular density of four human melanoma lines (A-07, D-12, R-18, U-25) grown intradermally in Balb/c nu/nu mice. VEGF expression, bFGF expression and expression of pimonidazole, a marker of hypoxic cells, were investigated by immunohistochemistry. An association between high VEGF and bFGF expression and high angiogenic potential was detected, suggesting an important role for VEGF/bFGF in the angiogenesis of melanomas. High VEGF/bFGF expression was also related to low hypoxic fraction and high vascular density. Thus, the constitutive, genetically determined level of VEGF was probably more important than hypoxia-induced upregulation in the angiogenesis of the melanoma xenografts.     

High expressions of CD105 and VEGF in early oral cancer predict potential cervical metastasis

BACKGROUND AND OBJECTIVES: Although elective neck dissection has become a popular treatment modality for early oral cancer among most head and neck surgeons, a large portion of patients revealed pathological N0 postoperatively. Our study is aimed to evaluate the expressions of the angiogenic factors, vascular endothelial growth factor (VEGF), and CD105 (endoglin) on the prediction of neck metastasis for clinical N0 patients in early oral cancer. PATIENTS AND METHODS: Between July 1996 and July 2005, the preoperative biopsy specimens among 176 patients who underwent surgical treatment for early oral cancer were retrieved for the immunohistochemical study for VEGF and CD105. RESULTS: High expressions of CD105 and VEGF significantly correlated with positive nodal metastasis, respectively (P<0.001). The expression of CD105 also significantly correlated with that of VEGF (P<0.001). Furthermore, the sensitivity and specificity for prediction of cervical metastasis by high expressions of CD105 versus VEGF were 81.8% and 97.7% versus 93.2% and 72%, respectively. Low expression of CD105 and VEGF also significantly correlated with higher survival rate, respectively (P<0.001). CONCLUSIONS: Besides the image studies, the expressions of both CD105 and VEGF could be useful to guide the elective treatment for clinical N0 neck in early oral cancer.     

Low pO2 and beta-estradiol induce VEGF in MCF-7 and MCF-7-5C cells: relationship to in vivo hypoxia

Previous work from this laboratory demonstrated that MCF-7 breast carcinoma cells grown in nude mice contained minimal hypoxia but that tamoxifen treatment of these tumors resulted in increased hypoxia (Evans S. et al., Cancer Research, 1997). These findings led to studies exploring the link between estrogen signaling and tumor oxygenation and determining the role of VEGF in this process. The stimulation of estrogen-dependent MCF-7 breast carcinoma cells in vitro with -estradiol resulted in a two-fold induction of VEGF mRNA and 1.3–2-fold increase in protein, similar to what was observed when these cells were exposed to 0.1% oxygen. Furthermore, the two stimuli given together had an additive effect on (increasing) VEGF expression, suggesting that the combination of hypoxia and estrogen may be important in upregulating VEGF in some breast cancers. Estrogen-independent MCF-7-5C cells, developed by growing MCF-7 cells in long-term culture in estrogen-free media, were also studied. Using EF5, a fluorinated 2-nitroimidazole which localizes to hypoxic cells, MCF-7-5C tumors grown in nude mice were found to contain lower pO₂ levels and more hypoxic regions than similarly grown MCF-7 tumors. We tested the hypothesis that this might be the result of defective expression of VEGF in MCF-7-5C cells in response to -estradiol and/or hypoxia. However, MCF-7-5C and MCF-7 cells showed a similar induction of VEGF in vitro in response to either -estradiol or hypoxia. Therefore, although these two cell lines grown as tumors have substantial differences in the presence and patterns of hypoxia, this could not be explained by a difference in VEGF induction.     

Expression of vascular endothelial growth factor in primary non-small cell lung carcinoma

TumorgrowthisdependentuponangiogenesisifitdevelopsfromminimalsizesuchasImm3toabiggersize.[]]Themechanismbywhichtumorsinduceangiogenesishavereceivedconsiderableattentioninrecentyears.Oneofthetumor-secretedangiogenesisfactors,vascularendothelialgrowthfactor(VEGF),appearstoplayanimportantroleintumorangiogenesis.["']VEGFexpressionhasbeendetectedinseveralhumantumors,includingglioblastoma,["']ovarian,[']breast,l']colorectal,[']kidney,[']bladderll()Iandgastriccarcinomas.lll]Blockingwithanti-VEG…     

EMMPRIN regulates tumor growth and metastasis by recruiting bone marrow-derived cells through paracrine signaling of SDF-1 and VEGF

EMMPRIN, a cell adhesion molecule highly expressed in a variety of tumors, is associated with poor prognosis in cancer patients. Mechanistically, EMMPRIN has been characterized to contribute to tumor development and progression by controlling the expression of MMPs and VEGF. In the present study, by using fluorescently labeled bone marrow-derived cells (BMDCs), we found that the down-regulation of EMMPRIN expression in cancer cells reduces tumor growth and metastasis, and is associated with the reduced recruitment of BMDCs. Further protein profiling studies suggest that EMMPRIN controls BMDC recruitment through regulating the secretion of soluble factors, notably, VEGF and SDF-1. We demonstrate that the expression and secretion of SDF-1 in tumor cells are regulated by EMMPRIN. This study reveals a novel mechanism by which EMMPRIN promotes tumor growth and metastasis by recruitment of BMDCs through controlling secretion and paracrine signaling of SDF-1 and VEGF.     

Vascular endothelial growth factor (VEGF) in leptomeningeal metastasis: diagnostic and prognostic value

This study examined the diagnostic and prognostic value of vascular endothelial growth factor (VEGF) levels in the cerebrospinal fluid (CSF) of 39 patients with leptomeningeal metastasis (LM). Vascular endothelial growth factor levels at diagnosis were significantly higher in patients with LM (median 359 pg ml(-1)) than in patients with other neurological diseases (median <25 pg ml(-1)). The specificity of VEGF levels above 250 pg ml(-1) for LM was high (98.3%), while the sensitivity was low (51.4%; 73% for VEGF values above 100 pg ml(-1)). In 49% of the LM patients, particularly with lymphoma or medulloblastoma, VEGF levels were below 250 pg ml(-1) and thus in the range of VEGF levels in other neurological diseases. Vascular endothelial growth factor levels correlated significantly with CSF lactate and albumin. Vascular endothelial growth factor levels mirrored the clinical course with a marked reduction in response to therapy and an increase at relapse in some patients who had serial CSF samples available. Multivariate Cox regression analysis showed VEGF below 100 pg ml(-1) (relative risk (RR)=4.24, P=0.0002) and age below 60 years (RR=2.5, P=0.004) to be associated with longer survival in LM. In conclusion, CSF VEGF levels in LM vary considerably. High VEGF levels have a very high specificity for LM and may help to establish the diagnosis. The role of VEGF as a predictor of outcome should be substantiated in prospective studies.     

IL-6 和 VEGF 在宫颈癌组织中的表达及其临床意义

目的：探讨IL-6在宫颈癌组织中表达的临床意义及其与VEGF的关系。方法：选择45例宫颈癌患者作为研究对象，另外同期选取23例因子宫肌瘤合并慢性宫颈炎行子宫全切的患者及20例因宫颈上皮内瘤样病变（CIN）行手术治疗患者的术后常规蜡块作为对照，采用免疫组织化学方法研究IL-6和VEGF在上述组织中的表达和临床意义探讨。结果：通过免疫组化法，IL-6和VEGF在宫颈癌和CIN组织中主要表达于癌细胞和异型细胞的胞膜和（或）胞质，IL-6在炎症宫颈组织中主要表达于上皮基底层细胞和上皮细胞的胞膜和（或）胞质。IL-6在炎症宫颈、CIN和宫颈癌中的阳性表达率分别为21．7％、55．O％和84．4％，阳性表达率显著升高（P〈0．05或P〈0．001）。VEGF在炎症宫颈、CIN和宫颈癌中阳性表达率分别为0％、50％和80％，阳性表达率也显著升高（P〈0．05或P〈0．01）。IL-6的表达与肿瘤大小、间质浸润深度和VEGF表达有关（P〈0．05）。结论：IL-6在宫颈癌组织中的表达是增高的，作用可能是促进了宫颈癌细胞的生长并且参与了血管生成过程。     

The role of endothelial cells in tumor invasion and metastasis

Summary Metastasis is one of the most devastating aspects of cancer. It is a complex multistep processes that results in spread of tumorigenic cells to secondary sites in various organs. The actual events that are involved in metastasis are the subject of several recent reviews [1–3].Upon growth of neoplastic cells beyond a certain mass (2 mm in diameter) an extensive vascularization through angiogenesis occurs. The new capillary network provides a supply of nutrients and gas exchange that allows further growth and development of the tumor mass. The network of the blood vessels also provides an entry site into the circulation for the neoplastic cells that detach from the tumor mass. Only a small percentage of circulating tumor cells (< 0.01%) survive travel in the circulation and arrest in the capillary beds of distant organs, extravasate and proliferate within the organ parenchyma producing a successful metastasis [1].Vasculature plays an important role in several steps of the metastatic process; 1) at the site of metastasis, vessels capture the cancer cell and provide the entry route into the secondary organ, and 2) through angiogenesis, vascular endothelial cells provide the supply of nutrients for the growth of the primary tumor mass and the route of intravasation. The lining of all blood vessels are covered with endothelial cells which play an active role in both processes. The metastatic properties of cancer cells have been extensively studied. Here, we will discuss the role of endothelial cells in the metastatic process with focus on their interaction with cancer cells at the site of extravasation.     

Vascular endothelial growth factor (VEGF) expression in operable gallbladder carcinomas

AIM: To investigate the angiogenic and prognostic role of vascular endothelial growth factor (VEGF) in operable gallbladder carcinomas. METHODS: Sixty patients with early gallbladder carcinomas, treated with surgery alone, were investigated immunohistochemically for the expression of VEGF, thymidine phosphorylase (TP) and new blood vessel formation. The results were correlated with clinico-pathological features and prognosis. RESULTS: An increased VEGF secretion in gallbladder carcinomas was significantly associated with increased angiogenesis but not with patients survival, although high angiogenesis did relate with poor prognosis. TP was also associated with angiogenesis, but only the combined VEGF/TP expression was associated with unfavourable survival. Histological grade was another independent factor of prognosis. CONCLUSION: Both VEGF and TP expression are associated with high rate of angiogenesis, a factor directly associated with prognosis. The combined expression of these angiogenic factors confer a particularly poor post-operative outcome, speculature.     

Tristetraprolin regulates expression of VEGF and tumorigenesis in human colon cancer

Tristetraprolin (TTP) is an AU‐rich element‐binding protein that regulates mRNA stability. Here, we report that TTP suppress the growth of human colon cancer cells both in vivo and in vitro by regulating of the expression of vascular endothelial growth factor (VEGF). TTP protein expression in human colonic tissues was markedly decreased in colonic adenocarcinoma compared with in normal mucosa and adenoma. VEGF expression was higher in colonic adenocarcinoma than in normal mucosa and adenoma. Specific inhibition of TTP expression by RNA‐interference increased the expression of VEGF in cultured human colon cancer cells, and TTP overexpression markedly decreased it. In addition, elevated expression of TTP decreased the expression level of luciferase linked to a 3′ terminal AU‐rich element (ARE) of VEGF mRNA. Colo320/TTP cells overexpressing TTP grew slowly in vitro and became tumors small in size when xenografted s.c into nude mice. These findings demonstrate that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells, suggesting that it can be used as novel therapeutic agent to treat colon cancer.     

丹参注射液对lewis肺癌小鼠移植瘤生长转移及VEGF表达的影响

目的：观察不同浓度丹参注射液对lewis肺癌小鼠移植瘤生长转移及肿瘤血管内皮细胞生长因子表达的影响。方法：将荷lewis肺癌的C57BL小鼠,分别腹腔注射不同浓度的丹参注射液[（5,10,20,40,80）g/kg]、环磷酰胺（CTX）、生理盐水,检测小鼠的肺部转移灶数和移植瘤的体积、重量及血管内皮生长因子（VEGF）的表达。结果：丹参各剂量组对荷瘤小鼠瘤重均有抑制作用,在实验范围内无明显的剂量依赖关系,与生理盐水组比较,丹参各剂量组差异无显著性（P〉0.05）。CTX组对荷瘤小鼠瘤重抑制较明显,与生理盐水组比较,差异有显著性（P〈0.05）。丹参各剂量组对荷瘤小鼠肿瘤体积有不同程度的抑制,但无明显的剂量依赖关系,与生理盐水组比较,丹参各组差异无显著性（P〉0.05）。CTX组对荷瘤小鼠肿瘤体积抑制较明显,与生理盐水组比较,差异有显著性（P〈0.01）。丹参各剂量组的肺转移灶数目均低于生理盐水组,但差异无显著性（P〉0.05）,CTX组肺转移灶数目低于生理盐水组,差异有显著性（P〈0.01）。丹参各剂量组对荷瘤小鼠移植瘤VEGF的表达与生理盐水组比较,差异无显著性（P〉0.05）,CTX组移植瘤VEGF的表达与生理盐水组比较,差异无显著性（P〉0.05）。丹参各剂量组对荷瘤小鼠脾、胸腺指数的影响与生理盐水组比较,差异无显著性（P〉0.05）,CTX组脾、胸腺指数与生理盐水组比较,差异无显著性（P〉0.05）。结论：不同浓度的丹参注射液对荷瘤小鼠的瘤重、瘤体积及肺部转移灶无明显的抑制或促进作用,对肿瘤转移相关因子VEGF的表达无影响。     

Neuropilins are multifunctional coreceptors involved in tumor initiation,growth, metastasis and immunity

The neuropilins (Nrps) are multifunctional proteins involved in development, immunity and cancer. Neuropilin-1 (Nrp1), or its homologue neuropilin-2 (Nrp2), are coreceptors that enhance responses to several growth factors (GFs) and other mediators. Nrps are coreceptors for the class 3 semaphorins (SEMA3), involved in axonal guidance, and several members of the vascular endothelial growth factor (VEGF) family. However, recent findings reveal they have a much broader spectrum of activity. They bind transforming growth factor β1 (TGF-β1) and its receptors, hepatocyte growth factor (HGF) and its receptor (cMet), platelet derived growth factor (PDGF) and its receptors, fibroblast growth factors (FGFs), and integrins. Nrps also promote Hedgehog signaling. These ligands and pathways are all relevant to angiogenesis and wound healing. In the immune system, the Nrps are expressed primarily by dendritic cells (DCs) and regulatory T cells (Tregs), and exert mainly inhibitory effects. In cancer, Nrps have been linked to a poor prognosis, which is consistent with their numerous interactions with ligands and receptors that promote tumor progression. We hypothesize that Nrps boost responses by capturing ligands, regulating GF receptor expression, endocytosis and recycling, and possibly also by signaling independently. Importantly, they promote epithelial-mesenchymal transition (EMT), and the survival of cancer stem cells. The recent finding that Nrps bind and internalize cell-penetrating peptides (CPPs) with arginine/lysine-rich C-terminal motifs (C-end rule; e.g., RXXR) is of interest. These CPPs can be coupled to large drugs for cancer therapy. Almost all studies have been preclinical, but findings suggest Nrps are excellent targets for anti-cancer drug development.