显性失活缺氧诱导因子-1通过下调VEGF表达促进宫颈癌细胞凋亡

将表达野生型缺氧诱导因子-1α (hypoxia inducible factor-1 α, HIF-1α)的重组质粒pcDNA3.1-full length HIF-1α,表达抑制型HIF-1α的重组质粒pcDNA3.1-dominant negative HIF-1α和空质粒pcDNA3.1稳定转染人宫颈癌SiHa细胞,研究HIF-1α对人宫颈癌SiHa细胞生物学行为的影响．采用免疫细胞化学法和Western 印迹检测HIF-1α与VEGF蛋白的表达;CoCl2化学缺氧法处理细胞,采用原位缺口末端标记(TUNEL)法检测细胞凋亡情况．结果显示,显性失活HIF-1α能下调VEGF蛋白的表达,促进细胞缺氧条件下的凋亡,这提示HIF-1α可能在宫颈癌的发生发展中起作用,利用显性失活HIF-1α转染抑制HIF-1α可望成为宫颈癌治疗基因治疗的又一新途径．     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression

We have previously demonstrated the roles of RhoA, Rac1, and Cdc42 in hypoxia-driven angiogenesis. However, the role of oncogenes in hypoxia signaling is poorly understood. Given the importance of Rho proteins in the hypoxic response, we hypothesized that Rho family members could act as mediators of hypoxic signal transduction. We investigated the cross-talk between hypoxia and oncogene-driven signal transduction pathways and explored the role of Rac1 on hypoxia-induced hypoxia-inducible factor (HIF)-1α and VEGF expression. Since the phosphatidylinositol 3'-kinase (PI3K) pathway is involved in signal transduction of many oncogenes, we explored the role of PI3K on Rac1-mediated expression of HIF-1α and VEGF in hypoxia. We showed that LY-294002, a PI3K inhibitor, suppressed HIF-1α and VEGF induction under hypoxic conditions by up to 50%. Activation of Rac1 resulted in an upregulation of hypoxia-induced HIF-1α expression, which was blocked by LY-294002. These data suggested that Rac1 is an intermediate in the PI3K-mediated induction of HIF-1α. Interestingly, there was a significant downregulation of the tumor suppressor genes p53 and von Hippel-Lindau tumor suppressor (VHL) in cells expressing a constitutively active form of Rac1. Rac1-mediated inhibition of p53 and VHL could therefore be implicated in the upregulation of HIF-1α expression.     

MicroRNA-22 regulates hypoxia signaling in colon cancer cells

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.     

Chronic hypoxia alters mitochondrial composition in human macrophages

Hypoxia inducible factors (HIFs) are important mediators of the cellular adaptive response during acute hypoxia. The role of HIF-1 and HIF-2 during prolonged periods of hypoxia, i.e. chronic hypoxia is less defined. Therefore, we used human THP-1 macrophages with a knockdown of either HIF-1α, HIF-2α, or both HIFα-subunits, incubated them for several days under hypoxia (1% O₂), and analyzed responses to hypoxia using 2D-DIGE coupled to MS/MS-analysis. Chronic hypoxia was defined as a time point when the early but transient accumulation of HIFα-subunits and mRNA expression of classical HIF target genes returned towards basal levels, with a new steady state that was constant from 72 h onwards. From roughly 800 spots, that were regulated comparing normoxia to chronic hypoxia, about 100 proteins were unambiguously assigned during MS/MS-analysis. Interestingly, a number of glycolytic proteins were up-regulated, while a number of inner mitochondrial membrane proteins were down-regulated independently of HIF-1α or HIF-2α. Chronic hypoxic conditions depleted the mitochondrial mass by autophagy, which occurred independently of HIF proteins. Macrophages tolerate periods of chronic hypoxia very well and adaptive responses occur, at least in part, independently of HIF-1α and/or HIF-2α and comprise mitophagy as a pathway of particular importance.     

Damage-associated molecular patterns in the pathogenesis of osteoarthritis: potentially novel therapeutic targets

Albendazole (ABZ) has an anti-tumor ability and inhibits HIF-1α activity. HIF-1α is associated with glycolysis and vascular endothelial cell growth factor (VEGF) expression, which plays an important role in cancer progression. These clues indicate that ABZ exerts an anti-cancer effect by regulating glycolysis and VEGF expression. The aim of this study is to clarify the effects of ABZ on non-small cell lung cancer (NSCLC) cells and explore the underlying molecular mechanisms. The expression levels of HIF-1α and VEGF were detected using western blot analysis, and the effect of ABZ on glycolysis was evaluated by measuring the relative activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) and detecting the production of lactate in A549 and H1299 cells. The results showed that ABZ decreased the expression levels of HIF-1α and VEGF and suppressed glycolysis in under hypoxia, but not normoxic condition. Inhibiting HIF-1α also suppressed glycolysis and VEGF expression. Additionally, ABZ inhibited the volume and weight, decreased the relative activities of HK, PK, and LDH, and reduced the levels of HIF-1α and VEGF of A549 xenografts in mouse models. In conclusion, ABZ inhibited growth of NSCLC cells by suppressing HIF-1α-dependent glycolysis and VEGF expression.     

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).     

VEGF、HIF-1α、Vasohibin在子宫内膜腺癌血管生成中的作用及与预后的关系

目的通过研究血管内皮生长因子(vascular endometrial growth factor,VEGF)、缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)和vasohibin在正常子宫内膜(normal endometrium,NE)和子宫内膜样腺癌(endom-etrioid adenocarcinoma,EA)组织中的表达,探讨上述指标与间质微血管密度(microvessel density,MVD)、EA临床病理特性及预后的关系。方法采用免疫组化Envision二步法检测105例EA和20例NE组织中VEGF、HIF-1α、vasohibin和MVD的表达。分析各指标相互之间及与EA临床病理特性和预后间的关系。结果(1)EA组中,VEGF、HIF-1α和MVD的表达明显高于NE组(P0.05),vasohibin的表达明显低于NE组(P0.01)。(2)VEGF、HIF-1α和MVD的表达与EA的手术病理分期相关(P0.05)。(3)在EA组,除vasohibin外,VEGF、HIF-1α和MVD三者的表达量两两间均呈正相关(P0.05)。(4)98例有完整随访资料患者的5年生存率为87.8%,VEGF、HIF-1α及vasohibin表达阳性组与阴性组5年生存率无显著性差异;MVD高表达组5年生存率明显低于MVD低表达组(P0.05)。结论异常增生的微血管与EA发生、发展及预后有关。VEGF、HIF-1α、vasohibin三者间动态平衡的失调促进了EA组织中的微血管生成,重建三者间的正常调节关系,刺激vasohibin的表达,可能是EA治疗的一个靶点。     

Hypoxia-increased RAGE expression regulates chemotaxis and pro-inflammatory cytokines release through nuclear translocation of NF-κ B and HIF1α in THP-1 cells

The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1?cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H₂O₂ or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1β, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1?cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H₂O₂. Moreover, SNP or H₂O₂ exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1?cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1β. Thus, hypoxia not only increases RAGE expression in THP-1?cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression.     

腺病毒介导的HIF-1α基因在缺血心肌中的表达

 研究腺病毒介导的低氧诱导因子=1α(HIF=1α)在急性心肌梗死(AMI)后缺血心肌中的表达变化.建立兔AMI模型,随机分为4组,分别心肌内注入腺病毒介导的HIF-1α基因(Ad-HIF-1α)、不含HIF-1α基因腺病毒颗粒(Ad--Blank)、HIF-1α核酶基因(Rz-HIF-1α),假手术组(Sham)为对照.RT-PCR和免疫组化方法分别检测HIF-1α及下游基因VEGF的mRNA和蛋白在不同时间点的表达.结果显示,AMI后1 d, HIF-1α、VEGF的mRNA和蛋白表达开始升高,7 d 达高峰,14 d、28 d逐渐下降,56 d时HIF-1αmRNA和蛋白无表达,而VEGF mRNA和蛋白仍有表达. 因此, 腺病毒介导的HIF-1α基因在缺血心肌能被有效转染并激活,其表达的时间窗为心肌缺血急性期至亚急性期.HIF-1α核酶基因能抑制HIF-1α及其下游基因的表达.     

Ascorbate antagonizes nickel ion to regulate JMJD1A expression in kidney cancer cells

Abnormal expression of histone demethylase Jumonji domain-containing protein 1A (JMJD1A) is associated with many kinds of cancers. JMJD1A is also a hypoxic response gene and its expression is regulated by hypoxia-inducible factor-1α (HIF-1α). In this study, we determined the role of JMJD1A in development and hypoxia pathway. We also measured the expression of JMJD1A and two hypoxia factors glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) in 786-0 and HEK293 cells treated with different concentrations of NiCl(2) (2.5-100 μM) for 24 h, and found that JMJD1A mRNA and protein were up-regulated with increased concentrations of NiCl(2). We then observed that ascorbate could retard the up-regulated effect of NiCl(2)-induced JMJD1A expression in a dose-dependent manner through decreasing the stability of HIF-1α protein. Immunohistochemical analysis further demonstrated ascorbate antagonized Ni(2+)-induced up-regulation of JMJD1A expression in 786-0, HEK293, and OS-RC-2 cells. These findings suggest that both Ni(2+) and ascorbate can regulate the expression of histone demethylase JMJD1A, which is important for cancer development or inhibition.     

Ascochlorin inhibits growth factor-induced HIF-1α activation and tumor-angiogenesis through the suppression of EGFR/ERK/p70S6K signaling pathway in human cervical carcinoma cells

Ascochlorin, a non-toxic prenylphenol compound derived from the fungus Ascochyta viciae, has been shown recently to have anti-cancer effects on various human cancer cells. However, the precise molecular mechanism of this anti-cancer activity remains to be elucidated. Here, we investigated the effects of ascochlorin on hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human epidermoid cervical carcinoma CaSki cells. Ascochlorin inhibited epidermal growth factor (EGF)-induced HIF-1α and VEGF expression through multiple potential mechanisms. First, ascochlorin selectively inhibited HIF-1α expression in response to EGF stimulation, but not in response to hypoxia (1% O(2)) or treatment with a transition metal (CoCl(2)). Second, ascochlorin inhibited EGF-induced ERK-1/2 activation but not AKT activation, both of which play essential roles in EGF-induced HIF-1α protein synthesis. Targeted inhibition of epidermal growth factor receptor (EGFR) expression using an EGFR-specific small interfering RNA (siRNA) diminished HIF-1α expression, which suggested that ascochlorin inhibits HIF-1α expression through suppression of EGFR activation. Finally, we showed that ascochlorin functionally abrogates in vivo tumor angiogenesis induced by EGF in a Matrigel plug assay. Our data suggest that ascochlorin inhibits EGF-mediated induction of HIF-1α expression in CaSki cells, providing a potentially new avenue of development of anti-cancer drugs that target tumor angiogenesis.