Mutant IkappaBalpha suppresses hypoxia-induced VEGF expression through downregulation of HIF-1alpha and COX-2 in human glioma cells

Our previous study demonstrated that mutant IkappaBalpha (IkappaBalphaM) could inhibit glioma angiogenesis and tumorigenesis through the downregulation of vascular endothelial growth factor (VEGF) and IL-8. However, the pathways involved in VEGF expression are not well understood. Growing evidence indicates that hypoxia-inducible factor-1alpha (HIF-1alpha) and cyclooxygenases-2 (COX-2) play important roles in this progression. In this study, we first examined the expressions of hypoxia-induced genes in human glioma cells transfected with IkappaBalphaM (IN500deltaM) or control plasmid (IN500delta) in vitro. We found that hypoxic stress induced the expressions of HIF-1alpha, COX-2, and VEGF, and that IkappaBalphaM completely suppressed these expressions in vitro. Next, we injected these glioma cells into nude mice. After 3 weeks, the mice were moved to a hypoxic chamber (10% oxygen) for 3, 12, 24, 48, 96, or 144 h. The expressions of HIF-1alpha, COX-2, and VEGF in vivo were then analyzed by Northern blot and immunohistochemistry. IkappaBalphaM suppressed the expression of hypoxia-induced HIF-1alpha gene in vivo, but hypoxic stress induced the expression of COX-2 after 72 h. VEGF induction followed after 96 h of hypoxia in IN500deltaM cells. These findings suggest that VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 and COX-2 genes, and IkappaBalphaM could inhibit VEGF expression through these two pathways. Thus, IkappaBalphaM is identified as a pivotal factor in angiogenesis and is a potential target for neoplasm therapy.     

Hypoxia enhances LPA-induced HIF-1alpha and VEGF expression: their inhibition by resveratrol

Lysophosphatidic acid (LPA) is a bioactive phospholipid that is involved in various cellular events, including tumor invasion and metastasis. In the present study, we investigated the effects of LPA and hypoxia on HIF-1alpha and VEGF expression, as well as the effect of resveratrol on LPA and hypoxia-induced HIF-1alpha and VEGF expression and human ovarian cancer cell migration. Our results show that LPA treatment under hypoxia increases HIF-1alpha protein level, which leads to increased expression of VEGF protein and mRNA. These increases in HIF-1alpha and VEGF expression are dramatically attenuated by resveratrol. The underlying mechanism of inhibition of HIF-1alpha expression by resveratrol seems to be associated with both inactivation of p42/p44 MAPK and p70S6K, as well as enhanced degradation of HIF-1alpha protein, resulting in profound decrease in VEGF expression and cell migration. Collectively, these results show that LPA under hypoxic condition enhances cell migration through the sequential induction of HIF-1alpha and VEGF, and that this enhancement is efficiently blocked by resveratrol.     

IL-27通过上调MIG和IP-10的表达抑制肿瘤血管形成

目的: 研究IL-27对肿瘤血管生成的抑制作用及其机制.方法:IL-27基因稳定转染的人食管癌细胞(Eca109/IL-27)接种于裸鼠,建立荷瘤裸鼠模型,观察肿瘤生长情况和裸鼠生存期.用ELISA法检测脾细胞IFN-γ的分泌水平;免疫组化法检测瘤组织中VEGF和CD34的表达,并通过CD34的水平计算微血管密度;用RT-PCR法检测肿瘤组织趋化因子IP-10、MIG mRNA的表达水平.结果:接种Eca109/IL-27细胞荷瘤小鼠的生存期较接种野生型Eca109细胞(未转染质粒)和Eca109/LXSN细胞(空载体质粒转染)小鼠的生存期明显延长(P<0.05).接种Eca109/IL-27细胞的裸鼠瘤组织中VEGF和CD34的表达水平显著性低于接种Eca109细胞和Eca109/LXSN细胞,微血管密度显著降低(均P<0.01).Eca109/IL-27组小鼠脾细胞产生较高水平的IFN-γ(P<0.05),趋化因子IP-10和MIG mRNA的表达水平也显著性高于接种Eca109细胞组和Eca109/LXSN细胞组(P<0.05).结论:IL-27在裸鼠体内通过上调IP-10和MIG表达抑制肿瘤血管生成,从而发挥抗肿瘤作用.     

PTHR1调控VEGF干预骨肉瘤组织血管形成及增殖的机制研究

目的：通过分析临床样本构建细胞转染调控模型,分析甲状旁腺激素1型受体（parathyroid hormone type 1 receptor,PTHR1）调控骨肉瘤血管生成和增殖的分子机制,为骨肉瘤的治疗提供新的临床思路。方法：分析2017年1月至2019年12月就诊于大连理工大学附属肿瘤医院（辽宁省肿瘤医院）与深圳市第二人民医院（深圳大学附属第一医院）,经组织病理学确认的四肢经典型骨肉瘤患者临床样本共50例,构建PTHR1沉默的骨肉瘤细胞模型,分析PTHR1在肿瘤组织样本中的差异表达,探索PTHR1表达水平与肿瘤增殖及患者生存率之间的相关性。通过体外实验下调PTHR1表达,分析其对血管生成的调控作用。结果：在骨肉瘤患者的临床样本中,PTHR1在肿瘤组织中的表达量显著高于瘤旁组织,且随着PTHR1表达量显著增高,患者肿瘤体积的增大,远期生存率下降。术后36个月生存率,PTHR1高表达组患者显著低于低表达组。在体外实验中,通过沉默PTHR1表达可以有效降低血管生成素血管内皮生长因子（vascular endothelial growth factor,VEGF）表达,抑制肿瘤细胞增殖,在...     

IL-27通过上调MIG和IP-10的表达抑制肿瘤血管形成

目的:研究IL27对肿瘤血管生成的抑制作用及其机制。方法： IL27基因稳定转染的人食管癌细胞（Eca109/IL27）接种于裸鼠,建立荷瘤裸鼠模型,观察肿瘤生长情况和裸鼠生存期。用ELISA法检测脾细胞IFNγ的分泌水平;免疫组化法检测瘤组织中VEGF和CD34的表达,并通过CD34的水平计算微血管密度;用RTPCR法检测肿瘤组织趋化因子IP10、MIG mRNA的表达水平。结果：接种Eca109/IL27细胞荷瘤小鼠的生存期较接种野生型Eca109细胞（未转染质粒）和Eca109/LXSN细胞（空载体质粒转染）小鼠的生存期明显延长（P<0.05）。接种Eca109/IL27细胞的裸鼠瘤组织中VEGF和CD34的表达水平显著性低于接种Eca109细胞和Eca109/LXSN细胞,微血管密度显著降低（均P<0.01）。Eca109/IL27组小鼠脾细胞产生较高水平的IFNγ（P<0.05）,趋化因子IP10和MIG mRNA的表达水平也显著性高于接种Eca109细胞组和Eca109/LXSN细胞组（P<0.05）。结论： IL27在裸鼠体内通过上调IP10和MIG表达抑制肿瘤血管生成,从而发挥抗肿瘤作用。     

双歧杆菌对兔VX2瘤HIF-1α和VEGF表达的影响

目的：探讨青春型双歧杆菌对兔VX2瘤组织HIF-1α、VEGF表达的影响。方法：建立兔荷VX2瘤模型20只，随机分成2组，每组10只。对照组经耳缘静脉注射生理盐水，实验组经耳缘静脉注射青春型双歧杆菌。处死动物，取出脏器和肿瘤组织粉碎后进行厌氧培养和革兰染色，观察双歧杆菌对肿瘤的靶向性；另取部分肿瘤组织H—E染色观察肿瘤组织的形态学变化；应用免疫组织化学方法分别检测肿瘤组织中HIF-1α、VEGF的表达。结果：青春型双歧杆菌能够靶向定植于肿瘤组织中；经双歧杆菌治疗后实验兔肿瘤组织坏死面积明显增加。免疫组化显示实验兔VX2瘤组织中HIF-1α、VEGF阳性表达细胞密度显著低于对照组（P〈0．05），且两组中HIF-1α与VEGF的表达呈显著正相关（r=0．86）。结论：青春型双歧杆菌能够在兔VX2瘤组织中靶向定植，且能下调兔VX2瘤中HIF-1α和VEGF的表达。     

Neuropilin-1及VEGF在胃癌组织中的表达与微血管生成的关系

  目的  探讨胃癌组织中神经纤毛蛋白-1（Neuropilin-1,NRP-1）、血管内皮生长因子（VEGF）的表达与微血管生成及胃癌生物学行为的关系和意义。  方法  应用RT-PCR法检测自2011年5月至2013年5月天津医科大学肿瘤医院72例手术切除的胃癌组织及癌旁正常组织中NRP-1 mRNA和VEGF mRNA的表达,免疫组织化学SP法检测组织中CD105标记的新生血管内皮细胞并测定微血管密度（MVD）,统计分析胃癌组织中NRP-1 mRNA、VEGF mRNA的表达与临床病理因素及MVD之间的关系。  结果  胃癌组织中NRP-1 mRNA、VEGF mRNA的表达及MVD计数均高于正常组织（P＜0.05）,且与肿瘤的浸润深度、淋巴结转移密切相关（P＜0.05）;胃癌组织中NRP-1 mRNA表达和VEGF mRNA表达呈正相关（r=0.58,P＜0.01）,NRP-1 mRNA的表达与MVD呈正相关（r= 0.52,P＜0.01）,VEGF mRNA的表达与MVD呈正相关（r=0.74,P＜0.01）。  结论  Neuropilin-1及VEGF的高表达促进了肿瘤微血管生成且与胃癌的进展密切相关,表达存在协同作用,两者的联合检测可在一定程度上判断胃癌侵袭性及进展,并对指导肿瘤抗血管治疗具有一定的价值。      

Alpha1-antitrypsin inhibits angiogenesis and tumor growth

Disturbances of the ratio between angiogenic inducers and inhibitors in tumor microenvironment are the driving force behind angiogenic switch critical for tumor progression. Angiogenic inhibitors may vary depending on organismal age and the tissue of origin. We showed that alpha(1)-antitrypsin (AAT), a serine protease inhibitor (serpin) is an inhibitor of angiogenesis, which induced apoptosis and inhibited chemotaxis of endothelial cells. S- and Z-type mutations that cause abnormal folding and defective serpin activity abrogated AAT antiangiogenic activity. Removal of the C-terminal reactive site loop had no effect on its angiostatic activity. Both native AAT and AAT truncated on C-terminus (AATDelta) inhibited neovascularization in the rat cornea and delayed the growth of subcutaneous tumors in mice. Treatment with native AAT and truncated AATDelta, but not control vehicle reduced tumor microvessel density, while increasing apoptosis within tumor endothelium. Comparative analysis of the human tumors and normal tissues of origin showed correlation between reduced local alpha(1)-antitrypsin expression and more aggressive tumor growth.     

TRAIL inhibits angiogenesis stimulated by VEGF expression in human glioblastoma cells

Tumour growth is tightly related to new blood vessel formation, tissue remodelling and invasiveness capacity. A number of tissular factors fuel the growth of glioblastoma multiforme, the most aggressive brain neoplasm. In fact, gene array analyses demonstrated that the proapoptotic cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) inhibited mRNA expression of VEGF, along with those of matrix metalloproteinase-2 (MMP-2), its inhibitor tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), as well as the tumour invasiveness-related gene secreted protein acid rich in cysteine (SPARC) in different human glioblastoma cell lines. Particularly, VEGF mRNA and protein expression and release from glioblastoma cells were also inhibited by TRAIL. The latter also exerted antimitogenic effects on human umbilical vein endothelial cells (HUVECs). With the same cells, TRAIL inhibited new vessel formation in the in vitro matrigel model, as well as it exerted powerful inhibition of blood vessel formation induced by an angiogenic cocktail administered in subcutaneous pellets in vivo in the C57 mouse. Moreover, the expression of MMP-2, its inhibitor TIMP-2 and the tumour invasiveness-related protein SPARC were effectively inhibited by TRAIL in glioblastoma cell lines. In conclusion, our data indicate that TRAIL inhibits the orchestra of factors contributing to glioblastoma biological aggressiveness. Thus, the TRAIL system could be regarded as a molecular target to exploit for innovative therapy of this type of tumour.     

Hypoxia-inducible factor-1alpha expression and angiogenesis in gastrointestinal stromal tumor of the stomach

Hypoxia inducible factor (HIF)-1 is reported to transactivate expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. The aim of this study was to elucidate the clinical significance of HIF-1alpha expression in gastrointestinal stromal tumors (GIST). Specimens obtained from 53 patients who underwent surgical resection for GIST of the stomach were used in this study. Specimens were examined immunohistochemically for HIF-1alpha, VEGF, and Ki-67 expression. Tumor microvessel density (MVD) was determined immunohistochemically with anti-CD31 antibody and was estimated by averaging the counts from three high-power fields in the area showing the greatest neovascularization. HIF-1alpha expression was detected in 17 (32.1%) of 53 lesions and was correlated significantly with tumor size, liver metastasis, VEGF expression, and MVD. Prognosis was significantly poorer in patients with tumors expressing HIF-1alpha than in patients with tumors lacking HIF-1alpha expression. HIF-1alpha may play a role in angiogenesis and tumor progression of GIST through regulation of VEGF.