干扰RNA沉默HIF-1α在缺氧状态下对骨肉瘤细胞VEGF表达的影响

背景与目的:研究表明H IF-1α是肿瘤适应低氧微环境、诱导血管新生的一个主要调控因子。本研究通过观察体外低氧培养条件下骨肉瘤细胞系SaOS-2中H IF-1α和VEGF的表达,探讨H IF-1α在骨肉瘤缺氧激活血管新生调控途径中的作用。方法:CoCl2化学缺氧法模拟肿瘤缺氧环境。半定量RT-PCR和免疫组化法分别检测不同缺氧时相中H IF-1α和VEGF在m RNA和蛋白水平的表达。构建针对H IF-1α的短发夹状小干扰RNA真核表达载体并转染SaOS-2细胞。免疫印迹沉淀观察转染后H IF-1α的基因沉默效果,RT-PCR和ELISA双抗体夹心法检测H IF-1α短基因沉默后SaOS-2细胞中VEGF的变化。结果:低氧条件下,SaOS-2细胞H IF-1αm RNA水平稳定,蛋白表达显著升高;而VEGF m RNA和蛋白的表达均显著升高。构建的H IF-1α短发夹状小干扰RNA表达载体转染SaOS-2细胞后能够显著下调H IF-1α基因的表达,同时VEGF基因的表达也受到明显抑制。结论:缺氧促使SaOS-2细胞H IF-1α在蛋白水平表达升高,H IF-1α通过转录激活VEGF的机制促进骨肉瘤缺氧状态下的血管新生。     

Colorectal carcinoma cell production of transforming growth factor beta decreases expression of endothelial cell vascular endothelial growth factor receptor 2

BACKGROUND:

Vascular endothelial growth factor (VEGF) signaling is a target for antiangiogenic cancer therapy. The authors have previously observed that up to 40% of vessels in colorectal carcinoma (CRC) tumors are negative for VEGF receptor 2 (VEGFR2) expression. Differential activity of transforming growth factor beta (TGF‐β) is a potential contributor to this receptor heterogeneity because TGF‐β contributes to both angiogenesis and CRC tumor progression.

METHODS:

The authors analyzed VEGFR2 expression by Western blotting, and TGF‐β expression in endothelial and CRC cell lines, respectively. In addition, they immunostained endothelial cells in CRC xenografts to find an association between VEGFR2 and TGF‐β levels or activity.

RESULTS:

In bovine aortic endothelial cells (BAECs), TGF‐β1 significantly repressed VEGFR2 protein in a time‐dependent and dose‐dependent fashion (P < .05). Serum‐free conditioned media from various malignant human CRC cell lines (HCT116, 379.2, Dks8, and DLD1) induced down‐regulation of VEGFR2 in BAECs. This effect was proportional to the total levels of TGF‐β1 and TGF‐β2 and was blocked by SB‐431542 and SD‐208, TGF‐β receptor I inhibitors. Immunofluorescence staining of subcutaneous mouse xenografts of HCT116, 379.2, Dks8, and SW480 cells revealed vessels with an inverse relationship between TGF‐β activity and VEGFR2 expression. Oxygen and bone morphogenetic protein 9 levels were shown to modulate TGF‐β–induced VEGFR2 down‐regulation.

CONCLUSIONS:

In combination with other factors, TGF‐β may contribute to the vascular heterogeneity in human colorectal tumors. Cancer 2011;. © 2011 American Cancer Society.     

Cannabinoids inhibit the vascular endothelial growth factor pathway in gliomas

Cannabinoids inhibit tumor angiogenesis in mice, but the mechanism of their antiangiogenic action is still unknown. Because the vascular endothelial growth factor (VEGF) pathway plays a critical role in tumor angiogenesis, here we studied whether cannabinoids affect it. As a first approach, cDNA array analysis showed that cannabinoid administration to mice bearing s.c. gliomas lowered the expression of various VEGF pathway-related genes. The use of other methods (ELISA, Western blotting, and confocal microscopy) provided additional evidence that cannabinoids depressed the VEGF pathway by decreasing the production of VEGF and the activation of VEGF receptor (VEGFR)-2, the most prominent VEGF receptor, in cultured glioma cells and in mouse gliomas. Cannabinoid-induced inhibition of VEGF production and VEGFR-2 activation was abrogated both in vitro and in vivo by pharmacological blockade of ceramide biosynthesis. These changes in the VEGF pathway were paralleled by changes in tumor size. Moreover, intratumoral administration of the cannabinoid Delta9-tetrahydrocannabinol to two patients with glioblastoma multiforme (grade IV astrocytoma) decreased VEGF levels and VEGFR-2 activation in the tumors. Because blockade of the VEGF pathway constitutes one of the most promising antitumoral approaches currently available, the present findings provide a novel pharmacological target for cannabinoid-based therapies.     

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。     

Cyclooxygenase-2 expression in advanced nasopharyngeal carcinoma--a prognostic evaluation and correlation with hypoxia inducible factor 1alpha and vascular endothelial growth factor

Cycloxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) can be induced by the Epstein-Barr virus oncoprotein latent membrane protein-1 (LMP-1) in nasopharyngeal cancer (NPC) cell lines. This study examined the prognostic relevance of COX-2 and its relationship with HIF-1alpha and VEGF expression in NPC biopsies. Primary tumor biopsies were obtained from 78 participants of a randomized trial who received radiotherapy (RT) with or without concurrent chemotherapy for locoregionally advanced NPC. These were analyzed for COX-2 expression and then correlated with age, sex, disease stage, treatment arm, survival and disease recurrence, VEGF and HIF-1alpha expression in a regression model. 83% of tumors expressed COX-2, 47% co-expressed COX-2 and VEGF, 38% co-expressed COX-2 and HIF-1alpha. On univariate analysis, COX-2 expression did not correlate with survival and recurrence, but moderate to high COX-2 expression was associated with advanced nodal stage (p=0.03). Although univariate analysis showed that COX-2-HIF-1alpha co-expression was associated with worse progression-free survival (p=0.046), time to local (p=0.004) and regional recurrence (p=0.007), multivariate analysis failed to confirm any correlation between COX-2-HIF-1alpha or COX-2-VEGF co-expression and survival or disease recurrence. Contrary to previous report, this study failed to demonstrate any prognostic significance of COX-2 expression alone or co-expression with HIF-1alpha or VEGF in advanced NPC.     

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.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

 目的　研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子（VEGF）及其受体（VEGFR）的影响。方法　酶联免疫吸附法检测非细胞毒性浓度（5、10、20 ng／ml）青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果　培养24、48 h后,无青蒿琥酯作用的 SHI-1细胞培养上清液VEGF质量浓度分别为（980.3±2.2）、（982.4±2.3）pg／ml,VEGFR-1表达率分别为（5.40±3.11）%和（4.45±2.85）%,VEGFR-2表达率分别为（13.90±2.26）%和（13.95±1.96）%。 5、10 、20 ng／ml青蒿琥酯作用24 h后,SHI-1细胞培养上清液VEGF质量浓度分别为（234.6±1.8）、（114.9±1.6）、（108.8±1.5）pg／ml,作用48 h后分别为（62.3±1.7）、（60.9±1.6）、（32.7±1.7）pg／ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义（均P＜0.05）。5、10 、20 ng／ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为（4.30±2.21）%、（4.20±1.37）%和（3.90±1.86）%,作用48 h后分别为（3.80±2.87）%、（3.60±1.73）%和（3.00±1.82）%,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义（均P＞0.05）;作用24 h后,SHI-1细胞VEGFR-2阳性率分别为（4.40±1.15）%、（3.10±0.68）%和（1.10±0.72）%,作用48 h后分别为（3.00±1.68）%、（2.20±0.93）%和（0.60±0.92）%,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞0.05）。结论　SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

Relationship between vascular endothelial growth factor expression and angiogenesis and cell proliferation of malignant gliomas in children

Thereismountingevidencethattumorangiogenesisisintimatelyrelatedtotumorgrowth,invasion,metastasisandprognosis.Tumorangiogenesisisregulatedbysomekindsofcytokinesintissues,suchasfibroblastgrowthfactor(FGF),transforminggrowthfactor(TGF),platelet-derivedgrowthfactor(PDGF)andvascularendothelialgrowthfactor(VEGF),amongwhich,VEGFisknowntobeparticularlyresponsiblefortheprocessofbraintumorangiogenesisVEGFinfluenceonbiologicalpropertiesofadultbraintumorhavebeenreportedinotherarticles.[1]Inthisstu…     

Restoration of wild-type p16 down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human gliomas.

Recent studies have indicated that the loss of p16 is a frequent event in the progression of malignant gliomas. The loss of p16 promotes the acquisition of malignant characteristics in gliomas, which are among the most angiogenic of all human tumors. High-grade gliomas are distinguished from low-grade gliomas by intense angiogenesis in addition to their frequent loss of p16. New therapeutic strategies aimed at inhibiting tumor angiogenesis on the basis of molecular mechanisms are theoretically attractive. Here we evaluate the effect of p16 gene replacement on the angiogenesis of gliomas. Infection with a recombinant replication-defective adenovirus vector containing the cDNA of wild-type p16 significantly reduced the expression of vascular endothelial growth factor, which is thought to be a pivotal mediator of tumor angiogenesis, in p16-deleted glioma cells. Restoring wild-type p16 expression into p16-deleted glioma cells markedly inhibited angiogenesis induced by tumor cells in vivo. Furthermore, wild-type p16 inhibited neovascularization more potently than did wild-type p53 transfer. These findings indicate that the p16 gene plays an important role in the regulation of glioma angiogenesis, suggesting a novel function of the p16 gene.