TRPV3 promotes the angiogenesis through HIF-1α-VEGF signaling pathway in A549 cells

BackgroundAngiogenesis is an essential physiological process in the growth and metastasis of primary tumors. Ca²⁺ signaling is crucial for tumor angiogenesis. The purpose of this study was to detect the potential role of Ca²⁺ permeable transient receptor potential vanilloid-3 (TRPV3) in the angiogenesis of non-small cell lung cancer (NSCLC).MethodsSmall interfering RNA was used to down-regulate TRPV3 expression in A549 cells. A laser scanning confocal microscope was used to examine intracellular calcium concentration ([Ca²⁺]i). Human umbilical vein endothelial cells (HUVECs) tube formation and migration assay, Western blot, MTT and ELISA were performed to detect the potential mechanisms of TRPV3 in tumor angiogenesis. A mouse tumor xenograft model was performed to expound the effects of TRPV3 on tumor cell growth.ResultsInhibition of TRPV3 reduced [Ca²⁺]i and protein expressions of VEGF and HIF-1α in A549 cells. Moreover, HIF-1α depletion decreased the secretion and expression of VEGF. Depletion of TRPV3 inhibited HUVECs proliferation, tube formation and migration induced by conditioned medium. And TRPV3 inhibition could decrease the volume of xenograft tumors and MVD of CD34⁺ cells. The expression levels of HIF-1α, VEGF and p-CaMKП in the xenograft tumors in RuR and siTRPV3 groups was reduced.ConclusionsTRPV3 calcium channel protein may play a key role in NSCLC angiogenesis. TRPV3 could promote the angiogenesis through HIF-1α-VEGF signaling pathway. Targeting TRPV3 channel protein by novel approaches would be useful for reversing NSCLC angiogenesis.     

Ginsenoside Rg3 inhibits HIF-1α and VEGF expression in patient with acute leukemia via inhibiting the activation of PI3K/Akt and ERK1/2 pathways

Aberrant angiogenesis is essential to the development and progression of leukemia. Ginsenoside Rg3 has been commonly used in anti-angiogenic therapy of solid tumors. This study aimed to investigate the anti-angiogenic effects of Rg3 in patients with acute leukemia. Bone marrow stromal cells derived from patients with acute leukemia were treated with Rg3 and the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α) was detected by RT-PCR and western blot analysis. The results showed that Rg3 inhibited VEGF and HIF-1α expression at both mRNA and protein levels in bone marrow stromal cells. In addition, Rg3 treatment led to reduced serum levels of HIF-1α and VEGF in patients with acute leukemia. Mechanistically, we demonstrated that Rg3 downregulated the phosphorylation of Akt and ERK1/2 in BMSCs. In conclusion, Rg3 exhibits anti-leukemia effect in part due to its anti-angiogenic activity via inhibiting PI3K/Akt and ERK1/2 pathways, which act to regulate the expression of HIF-1α and VEGF.     

Inducible expression of endothelial PAS domain protein-1 by hypoxia in human lung adenocarcinoma A549 cells. Role of Src family kinases-dependent pathway.

Hypoxia is a potent inducer of tumor angiogenesis, the process of which is mostly mediated by induction of vascular endothelial growth factor (VEGF). In this study, we investigated the effect of hypoxia on the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and endothelial PAS domain protein-1 (EPAS1). These two similar but distinct basic helix-loop-helix-PAS proteins have been postulated to activate VEGF expression in response to hypoxia. We showed that EPAS1, but not HIF-1alpha, is abundantly expressed in human lung adenocarcinoma A549 cells. Exposure of cultured A549 cells to hypoxia increased EPAS1 mRNA and protein levels. A specific inhibitor for Src family kinases, PP1, abolished the hypoxia-induced expression of EPAS1. Transient transfection assays revealed that forced expression of EPAS1 increased the reporter gene activity driven by EPAS1 promoter as well as by VEGF promoter. Finally, overexpression of EPAS1 by infection of adenoviral vector expressing EPAS1 cDNA evidently induced the endogenous EPAS1 gene expression. Together, these data demonstrate Src family kinases mediate the hypoxia-mediated EPAS1 gene expression, which in turn positively autoregulates its own expression. Given an EPAS1 as a potent activator of the VEGF gene, these findings will provide a novel insight into the mechanisms underlying the enhancement of growth property of EPAS1-expressing tumor cells under the hypoxic environment.     

褐藻糖胶对RPMI8226细胞血管生成的影响

 目的: 探讨褐藻糖胶对多发性骨髓瘤RPMI 8226细胞血管生成的影响及可能的作用机制。方法: 体外培养多发性骨髓瘤RPMI 8226细胞及人血管内皮细胞EA.hy 926细胞,采用MTT法检测褐藻糖胶对RPMI 8226细胞活力的影响;流式细胞术检测细胞周期及凋亡率;ELISA法检测褐藻糖胶处理RPMI 8226细胞后的培养液上清中VEGF的含量;小管形成实验观察褐藻糖胶处理RPMI 8226细胞后的培养液上清对EA.hy 926细胞诱导小管形成能力的影响;Western blot检测HIF-1α、VEGF、p-AKT和p-ERK1/2的蛋白水平。结果: 褐藻糖胶对RPMI 8226细胞活力的抑制作用呈浓度及时间依赖性;褐藻糖胶作用RPMI 8226细胞72 h后,细胞周期被阻滞于G₁期,细胞凋亡率呈浓度依赖性明显增高,各组均高于对照组(P<0.05);ELISA法结果显示褐藻糖胶处理72 h后的细胞培养液上清中VEGF的含量明显减少,与对照组相比,处理组上清中VEGF的含量下降(P<0.05);内皮细胞形成小管数目与面积随着褐藻糖胶的浓度升高而减小,100 mg/L 组差异显著(P<0.05);Western blot结果显示HIF-1α、VEGF、p-AKT和p-ERK1/2的蛋白水平与褐藻糖胶浓度呈负相关(P<0.05)。结论: 褐藻糖胶减少骨髓瘤细胞自分泌VEGF,减弱血管内皮细胞形成小管的能力,降低HIF-1α和VEGF的蛋白水平,这可能与抑制AKT和ERK1/2的磷酸化有关。     

Actin-sequestering protein,thymosin beta-4, is a novel hypoxia responsive regulator

Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.     

人非小细胞肺癌中HIF-1α的表达与血管生成和细胞凋亡的关系及意义

目的 探讨缺氧诱导因子1α(HIF-1α)在非小细胞肺癌(NSCLC)中的表达及其与血管生成和细胞凋亡的关系,评估它在NSCLC发生发展过程中的作用.方法 采用免疫组织化学的方法检测HIF-1α在正常肺组织、良性病变、癌前病变、原位癌、非小细胞肺癌及肺癌转移淋巴结中的表达,及血管内皮生长因子(VEGF)在NSCLC中的表达;采用TUNEL法检测部分NSCLC中细胞凋亡指数AI.结果 HIF-1α在支气管黏膜非典型增生、原位癌、NSCLC及肺癌转移淋巴结中的阳性表达率相似,总阳性表达率为40.0%(68/170),其在正常及良性病变中的总阳性表达率为6.0%(4/67),两组间有显著差异(P＜0.01).HIF-1α在NSCLC中的表达与各项临床病理特征均无明显相关性,仅与患者术后生存时间明显正相关(P＜0.01).VEGF在NSCLC中的表达与HIF-1α的表达呈正相关(P＜0.01),与患者预后呈负相关(P=0.027).凋亡指数(AI)在各组织类型和各分化程度的Ⅰ期肺癌中无明显差异,但生存期≥5年的肺癌患者AI明显高于生存期＜5年的患者(P=0.004).AI与HIF-1α的表达明显正相关(P=0.004).结论 HIF-1α在非小细胞肺癌组织中的表达较正常及良性病变肺组织明显上调,其与肿瘤血管生成及细胞凋亡均密切相关,在NSCLC生长中的作用具有多向性.HIF-1α可以作为评估患者预后的重要指标.     

HIF-1α and VEGF expression correlates with thrombus remodeling in cases of intravascular papillary endothelial hyperplasia

Intravascular papillary endothelial hyperplasia (IPEH) is histopathologically characterized by endothelium-lined papillary structures encircling an acellular fibrin core. The process of IPEH pathogenesis is unclear. The purpose of our study was to identify histopathological and immunohistochemical characteristics of IPEH to better understand the pathogenesis of this disease. After reviewing microscopic and medical records from Kyungpook National University Hospital, we selected 16 cases of IPEH. Masson’s trichrome and immunohistochemical staining as well as hematoxylin-eosin staining for 16 cases of IPH were performed. Immunohistochemical studies included CD31, CD68, mast cell tryptase, hypoxia-inducible factor-1 (HIF-1α), and vascular endothelial growth factor (VEGF). Sections from all our cases showed three distinct histological regions including a papillary portion with hyalinized fibrous or fibroblastic cores, an area containing an unorganized thrombus, and organization area with an ingrowth of endothelial cells, myofibroblasts, and fibroblasts. In the organization area, HIF-1α-positive cells were identified in the loose connective tissue. Endothelial cells forming vascular channels were negative for HIF-1α while VEGF was highly expressed in both interstitial mononuclear and endothelial cells. In the papillary portion, the cellular cores were strongly positive for both HIF-1α and VEGF, but the acellular cores were negative. Our investigation confirmed that IPEH is a reactive lesion that incidentally arises during the organization process of older thrombi. It was also found that HIF-1α and VEGF expression was dependent on the thrombus remodeling stage in cases of IPEH.     

HIF-1α介导了间歇低氧对人肺腺癌A549细胞体外生物学行为的影响

目的:探讨缺氧诱导因子1α(HIF-1α)是否介导间歇低氧对人肺腺癌A549细胞体外活力、凋亡以及侵袭的影响。方法:采用体外转染方法将特异性针对HIF-1α的siRNA导入A549细胞,在间歇低氧条件下培养后通过real-time PCR和Western blot法检测HIF-1α及其下游Bcl-2、Bax、P53、P21、VEGF的mRNA和蛋白表达;通过MTT法和流式细胞术分别检测A549细胞的活力、凋亡及细胞周期;通过Transwell小室检测A549细胞的侵袭能力。结果:未转染HIF-1α-siRNA的A549细胞[间歇低氧空白对照组(IHC组)、间歇低氧空载体对照组(IHE组)及间歇低氧阴性对照组(IHN组)]经间歇低氧干预后的HIF-1α、Bcl-2及VEGF表达均明显高于常氧对照组(RA组),Bax及P21表达均明显低于RA组(P0.05),而转染HIF-1α-siRNA的A549细胞[间歇低氧siRNA组(IHS组)]较IHC组、IHE组及IHN组经间歇低氧干预后的HIF-1α、BCL-2及VEGF表达均明显下调,Bax及P21表达较均明显上调(P0.05);所有间歇低氧组A549细胞的P53表达均较RA组升高(P0.05),但各间歇低氧组之间无显著性差异。IHC组、IHE组及IHN组A549细胞经间歇低氧干预后较RA组的细胞活力增强,凋亡率下降,侵袭力增强(P0.05),而IHS组A549细胞经间歇低氧干预后细胞周期被阻滞于G1期,较未转染组的细胞活力下降,凋亡增加,侵袭能力下降(P0.05)。结论:间歇低氧可通过HIF-1α途径调控其下游基因表达进而促进A549细胞的活力和转移;通过RNA干扰技术造成HIF-1α基因沉默可以抑制间歇低氧引起的A549细胞生长和转移。     

Hypoxia-inducible factor-1alpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis

Angiogenesis progresses together with fibrogenesis during chronic liver injury. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of homeostasis, plays a pivotal role in hypoxia-induced angiogenesis through its regulation of vascular endothelial growth factor (VEGF). The association between hypoxia, angiogenesis and VEGF expression has been demonstrated in experimental cirrhosis. However, expression of HIF-1alpha has yet to be reported. The aim of this study was to investigate the significance of HIF-1alpha expression during experimental liver fibrosis and the relationships between HIF-1alpha expression, VEGF expression and angiogenesis. Cirrhosis was induced in male Wistar rats by intraperitoneal administration of diethyl nitrosamine (DEN) (100 mg/kg, once a week). The serial sections from liver tissues were stained with anti-HIF-1alpha, anti-VEGF and anti-CD34 antibodies before being measured by light microscopy. Our results showed that HIF-1alpha expression gradually increases according to the severity of fibrosis (p<0.01). Moreover, its expression was found to be correlated with angiogenesis (r=0.916) and VEGF expression (r=0.969). The present study demonstrates that HIF-1alpha might have a role in the development of angiogenesis via regulation of VEGF during experimental liver fibrogenesis and suggests that this factor could be a potential target in the manipulation of angiogenesis in chronic inflammatory diseases of the liver.     

沉默HIF-1α基因表达对大鼠肝癌细胞增殖的影响

 目的： 探讨沉默缺氧诱导因子　1α（HIF-1α）基因表达对缺氧状态下肝癌细胞增殖的影响。方法：选用大鼠CBRH-7919肝癌细胞株作为研究对象,利用氯化钴（CoCl2）建立缺氧模型。制备3组特异性较强的HIF-1α siRNA-脂质体复合物,转染于肝癌细胞。利用real-time RT-PCR、Western blotting等方法分别检测肝癌细胞HIF-1α、血管内皮生长因子(VEGF)、p21和cyclin D1在mRNA和（或）蛋白水平的表达。MTT及BrdU 掺入实验检测细胞增殖的变化。结果：缺氧条件下,肝癌细胞的HIF-1α和VEGF mRNA及蛋白表达显著增多（P<0.05）。HIF-1α基因表达沉默后,HIF-1α、VEGF及cyclin D1 mRNA和（或）蛋白表达明显减少（P<0.05）,p21蛋白表达明显增加（P<0.05）。HIF-1α siRNA转染组的BrdU阳性细胞比例明显少于对照组(P<0.05)。结论：沉默HIF-1α基因表达对缺氧状态下肝癌细胞的增殖有显著抑制作用。     

紫苏醇对肺癌A549 细胞增殖和侵袭的抑制作用机制的研究

目的:探讨紫苏醇(Perillyl alcohol,PA)对肺癌A549 细胞增殖和侵袭的抑制作用机制,并阐明其对肿瘤血管生成信号的影响。方法:不同浓度PA 和厄洛替尼加入到A549 细胞中,利用溴化3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑(MTT)法测定药物组对A549 细胞的抑制作用,Transwell 法检测PA 对A549 细胞侵袭的抑制作用,采用间接荧光标记法测定细胞内活性氧(ROS)水平的变化;分光光度法检测PA 对细胞凋亡蛋白Caspase-3 活性的影响,Western blot 法检测A549 中VEGF、HIF-1 及COX-2 的表达,凝胶迁移或电泳迁移率实验测定A549 细胞中NF-κB 活性。结果:与空白对照组比较,随着浓度的增加(10、50、100 g/ ml),PA 和厄洛替尼对A549 细胞生长的抑制率在不断地增加,差异均有统计学意义(P<0.05),A549 细胞侵袭能力呈现不断下降的趋势,差异均有统计学意义(P<0.05),A549 细胞内活性氧水平随厄洛替尼浓度的增加变化不大,而ROS 水平随着PA 的浓度的增加而增加,在100 g/ ml 浓度的PA 下引起的ROS 百分率达到了(80.43±6.92)%,差异均有统计学意义(P<0.05)。细胞活力检测结果显示,随着PA 和厄洛替尼浓度的增加和作用时间的延长,A549 细胞中凋亡蛋白Caspase-3 活性明显增加(P<0.05),随着紫苏醇浓度的增加,COX-2、VEGF 和HIF-1 的表达呈不断降低的趋势,EMSA 检测结果显示,随着PA 浓度的增加,NF-κB 的条带面积不断减少。结论:PA 可能参与并促进了ROS 的生成和Caspase-3 活性增加,最终诱导A549 细胞的凋亡,PA 可能通过降低NF-κB 的表达,进而诱导COX-2、VEGF 等的表达减少,使血管生成滞后,能够有效地使细胞的穿透能力下降和凋亡发生。