Expression of hypoxia-inducible factor (HIF)-1alpha is associated with vascular endothelial growth factor expression and tumour angiogenesis in human oesophageal squamous cell carcinoma

The purpose of this study was to examine the relationship between hypoxia inducible factor (HIF)-1alpha expression, vascular endothelial growth factor (VEGF) expression, and tumour vascularity in squamous cell carcinoma of the oesophagus. Expression of HIF-1alpha and VEGF was examined in two oesophageal squamous cell carcinoma cell lines (TE2, TE3) and 82 archival surgical specimens of human oesophageal squamous cell carcinoma tissue. In both cell lines, the levels of HIF-1alpha protein and VEGF mRNA were increased under hypoxic conditions. Thirty-two of the 82 (39%) tumour specimens showed high levels of HIF-1alpha immunoreactivity in the nuclei and/or cytoplasm of cancer cells. HIF-1alpha expression correlated significantly with venous invasion, VEGF expression, and microvessel density. Among the 47 patients who did not receive pre-operative chemotherapy, the outcome of those with high HIF-1alpha-expressing tumours was significantly poorer than that of those with low HIF-1alpha-expressing tumours. These results suggest that HIF-1alpha and VEGF expression are important determinants of survival in squamous cell carcinoma of the oesophagus.     

大肠腺瘤和腺癌组织中缺氧诱导因子-1α的表达及其与VEGF、微血管密度的关系

背景与目的:缺氧诱导因子-1α(hypoxia-inducible factor 1 alpha,HIF-1α)是肿瘤细胞适应缺氧而产生的一种核转录因子,在促进肿瘤新生血管生成中起重要作用。本研究旨在探讨大肠腺瘤和腺癌组织中HIF-1α的表达及其与血管内皮生长因子(vascular endothelial growth factor,VEGF)、微血管密度(microvessel density,MVD)的关系。方法:采用原位杂交技术检测HIF-1α mRNA,应用免疫组织化学方法检测VEGF蛋白的表达,用CD34单克隆抗体标记血管内皮细胞并计数MVD。结果:大肠腺癌组织HIF-1α mRNA阳性表达率为67.8％(42/62),腺瘤组织为44.4％(8/18)。腺癌组织从Dukes’A期到Dukes’C+D期HIF-lα mRNA表达阳性率不断增加(P<0.05)。HIF-1α mRNA表达平均阳性率为:腺瘤44.4％;腺癌Dukes’A期41.2％,Dukes’B期72.2％,Dukes’C+D期81.5％。腺癌组VEGF阳性表达率高于腺瘤组(59.7％ vs 33.3％,P<0.005).HIF-1α表达与VEGF呈正相关(r_s=0.768,P<0.01),与MVD呈正相关(r_s=0.683,P<0.05)。结论:HIF-1α及其靶基因 VEGF的过度表达与肿瘤新生血管形成呈正相关,这在大肠腺瘤癌变及大肠腺癌发展过程中可能起重要作用。     

trans-3,4,5'-Trihydroxystibene inhibits hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in human ovarian cancer cells.

trans-3,4,5'-Trihydroxystibene (resveratrol) is a natural product commonly found in the human diet and has been shown recently to have anticancer effects on various human cancer cells. However, the molecular basis for its anticancer action remains to be elucidated. In this study, we investigated the effect of resveratrol on hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression in human ovarian cancer cells A2780/CP70 and OVCAR-3. We found that although resveratrol did not affect HIF-1alpha mRNA levels, it did dramatically inhibit both basal-level and growth factor-induced HIF-1alpha protein expression in the cells. Resveratrol also greatly inhibited VEGF expression. Mechanistically, we demonstrated that resveratrol inhibited HIF-1alpha and VEGF expression through multiple mechanisms. First, resveratrol inhibited AKT and mitogen-activated protein kinase activation, which played a partial role in the down-regulation of HIF-1alpha expression. Second, resveratrol inhibited insulin-like growth factor 1-induced HIF-1alpha expression through the inhibition of protein translational regulators, including M(r) 70,000 ribosomal protein S6 kinase 1, S6 ribosomal protein, eukaryotic initiation factor 4E-binding protein 1, and eukaryotic initiation factor 4E. Finally, we showed that resveratrol substantially induced HIF-1alpha protein degradation through the proteasome pathway. Our data suggested that resveratrol may inhibit human ovarian cancer progression and angiogenesis by inhibiting HIF-1alpha and VEGF expression and thus provide a novel potential mechanism for the anticancer action of resveratrol.     

Brain-derived neurotrophic factor activation of TrkB induces vascular endothelial growth factor expression via hypoxia-inducible factor-1alpha in neuroblastoma cells

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Recently, we have shown that insulin-like growth factor-I and serum-derived growth factors stimulate VEGF expression in neuroblastoma cells via induction of hypoxia-inducible factor-1alpha (HIF-1alpha). Because another marker of poor prognosis in neuroblastoma tumors is high expression of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, TrkB, we sought to evaluate the involvement of BDNF and TrkB in the regulation of VEGF expression. VEGF mRNA levels in neuroblastoma cells cultured in serum-free media increased after 8 to 16 hours in BDNF. BDNF induced increases in VEGF and HIF-1alpha protein, whereas HIF-1beta levels were unaffected. BDNF induced a 2- to 4-fold increase in VEGF promoter activity, which could be abrogated if the hypoxia response element in the VEGF promoter was mutated. Transfection of HIF-1alpha small interfering RNA blocked BDNF-stimulated increases in VEGF promoter activity and VEGF protein expression. The BDNF-stimulated increases in HIF-1alpha and VEGF expression required TrkB tyrosine kinase activity and were completely blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. These data indicate that BDNF plays a role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to BDNF/TrkB, PI3K, mTOR signal transduction pathways, and/or HIF-1alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.     

Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression

In order to verify whether tenascin-C (TN-C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375-GFP), implanted subcutaneously into BALB/cA nude (WT) and TN-C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375-GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell-cell interactions. Both the content of TN-C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375-GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN-C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN-C expression of A375-GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN-C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN-C. These results clearly indicated that the expressions of both TN-C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN-C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development.     

Energy intake and prostate tumor growth, angiogenesis, and vascular endothelial growth factor expression

BACKGROUND: A sedentary lifestyle coupled with excessive energy intake is speculated to be a factor associated with increased incidence of prostate cancer. We have investigated the effects of energy intake on prostate tumor growth in experimental animals. METHODS: Two transplantable prostate tumor models, i.e., the androgen-dependent Dunning R3327-H adenocarcinoma in rats and the androgen-sensitive LNCaP human carcinoma in severe combined immunodeficient mice, were studied. R3327-H tumor growth and relevant tumor biomarkers (proliferation index, apoptosis [programmed cell death], microvessel density, and vascular endothelial growth factor [VEGF] expression) were compared in ad libitum fed control rats, ad libitum fed castrated rats, and groups restricted in energy intake by 20% or 40%. A second set of experiments involving both tumor models examined tumor growth in ad libitum fed rats or in animals whose energy intake was restricted by 30% using three different methods, i.e., total diet restriction, carbohydrate restriction, or lipid restriction. All P values are two-sided. RESULTS: R3327-H tumors were smaller in energy-restricted or castrated rats than in control rats (P<.001). Tumors from energy-restricted rats exhibited changes in tumor architecture characterized by increased stroma and more homogeneous and smaller glands. In castrated rats, the tumor proliferation index was reduced (P<.0001), whereas apoptosis was increased in both energy-restricted (P<.001) and castrated (P<.001) rats. Tumor microvessel density and VEGF expression were reduced by energy restriction and castration (P<.003 versus control). Restriction of energy intake by reduction of carbohydrate intake, lipid intake, or total diet produced a similar inhibition of growth of R3327-H or LNCaP tumors. These effects were associated with reduced circulating insulin-like growth factor-I. CONCLUSIONS: Our observations are consistent with the hypothesis that energy restriction reduces prostate tumor growth by inhibiting tumor angiogenesis. Furthermore, dietary fat concentration does not influence prostate tumor growth when energy intake is reduced.     

Molecular mediators of tumor angiogenesis: enhanced expression and activation of vascular endothelial growth factor receptor KDR in primary breast cancer.

The progression of breast cancer growth and its ability to metastasize are associated with the process of angiogenesis. In this study, we examined the protein expression of vascular endothelial growth factor (VEGF) and its specific and functional receptor KDR in human breast tissue. We investigated a total of 13 mammary carcinomas, 3 fibroadenomas, 5 specimens with fibrocystic breast disease as well as normal (adjacent to malignant) breast tissue using immunohistochemistry and Western blot analysis. In all carcinomas examined, functional KDR protein was present independent of tumor type, tumor stage and histological grade as demonstrated by tyrosine phosphorylation analysis of KDR. When malignant tissues were compared with their neighboring non-neoplastic regions, activated KDR was found to be expressed to a much higher extent within the malignant tissue samples. In fibroadenomas, KDR was barely detectable, whereas in fibrocystic breast disease KDR expression was variable. Immunostaining of KDR was localized to endothelium and epithelium of mammary ducts in malignant and benign breast tissue, while VEGF immunoreactivity was primarily found in the endothelium and also in tumor cells and macrophages. Our data demonstrate that KDR activation is enhanced in breast cancer in vivo and emphasize the functional role of VEGF and KDR in the development of malignant breast disease.     

Overexpression of human papillomavirus type 16 oncoproteins enhances hypoxia-inducible factor 1 alpha protein accumulation and vascular endothelial growth factor expression in human cervical carcinoma cells.

PURPOSE: Human papillomavirus (HPV)-16 oncoproteins, E6 and E7, are associated with enhanced tumor angiogenesis in human cervical cancers. The purpose of this study was (a) to investigate whether expression of HPV-16 E6 and E7 oncoproteins induces hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor expression in cervical cancer cells; and (b) to assess the effect of resveratrol on 16 E6- and E7-induced HIF-1 alpha and VEGF gene expression. EXPERIMENTAL DESIGN: Human cervical cancer cell lines C-33A and HeLa were transiently cotransfected with pSG5-HPV-16 E6 or 16 E7 constructs along with HIF-1 alpha small interfering RNA (siRNA) or nonspecific siRNA. The expression of HIF-1 alpha/VEGF was measured using real-time PCR, Western blot analysis, or ELISA. The in vitro angiogenic activity induced by 16 E6- and E7-transfected cells was examined. The effect of resveratrol on oncoprotein-induced HIF-1 alpha/VEGF expression and in vitro angiogenesis was investigated. RESULTS: HPV-16 E6- and E7-transfected cervical cancer cells express increased HIF-1 alpha protein and VEGF expression. These stimulatory effects were abrogated by cotransfection with either HIF-1 alpha siRNA or treatment with resveratrol. Blocking extracellular signal-regulated kinase 1/2 (ERK 1/2) and phosphoinositide-3-kinase by PD98059 and LY294002, respectively, abolished 16 E6- and E7-induced HIF-1 alpha and VEGF expression. Functionally, we showed that HPV-16 E6- and E7-transfected cervical cancer cells stimulated in vitro capillary or tubule formation, and these angiogenic effects could be abolished either by cotransfection with HIF-1 alpha siRNA or by treatment with resveratrol. CONCLUSION: HPV-16 oncoproteins contribute to enhanced angiogenesis in cervical cancer cells via HIF-1 alpha-dependent VEGF expression. Resveratrol suppresses 16 E6- and E7-induced HIF-1 alpha-mediated angiogenic activity and, thus, is a promising chemotherapeutic agent for human cervical cancer.     

Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice

Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)-specific angiogenesis are all enhanced in beta(3)-integrin-null mice. Furthermore, endothelial cells isolated from beta(3)-null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that beta(3)-integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in beta(3)-null mice. Moreover, beta(3)-null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation, beta(3)-null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild-type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in beta(3)-null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in beta(3)-integrin-deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth observed in these mice in vivo.     

Placenta growth factor overexpression inhibits tumor growth, angiogenesis, and metastasis by depleting vascular endothelial growth factor homodimers in orthotopic mouse models

The role of placenta growth factor (PlGF) in pathologic angiogenesis is controversial. The effects of PlGF on growth, angiogenesis, and metastasis from orthotopic tumors are not known. To this end, we stably transfected three human cancer cell lines (A549 lung, HCT116 colon, and U87-MG glioblastoma) with human plgf-2 full-length cDNA. Overexpression of PlGF did not affect tumor cell proliferation or migration in vitro. The growth of PlGF-overexpressing tumors grown orthotopically or ectopically was impaired in all three tumor models. This decrease in tumor growth correlated with a decrease in tumor angiogenesis. The PlGF-overexpressing tumors had decreased vessel density and increased vessel diameter, but vessel permeability was not different from the parental tumors. Tumors overexpressing PlGF exhibited higher levels of PlGF homodimers and PlGF/vascular endothelial growth factor (VEGF) heterodimers but decreased levels of VEGF homodimers. Our study shows that PlGF overexpression decreases VEGF homodimer formation and inhibits tumor progression.