Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O(2) level, which is regulated by many physiological functions. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1alpha involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1alpha (HIF-1alpha). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1alpha in microglia.     

Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells

Hypoxia appears to induce a program which shifts the cellular phenotype toward an increase in extracellular adenosine. Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. Since in gliomas there is a strong correlation between HIF-1alpha expression, tumor grade and tumor vascularization, the aim of this study was to investigate whether adenosine may regulate HIF-1 in human glioblastoma cell lines. The results indicate that in the human hypoxic A172 and U87MG glioblastoma cell lines adenosine up-regulates HIF-1alpha protein expression via the A(3) receptor subtype. In particular, we investigated the effect of A(3) receptor antagonists on HIF-1 and vascular endothelial growth factor (VEGF) expression. We found that A(3) antagonists inhibit adenosine-induced HIF-1alpha and VEGF protein accumulation in the hypoxic cells. Investigations in the molecular mechanism showed that A(3) receptor stimulation activates p44/p42 and p38 MAPKs that are required for A(3)-induced increase of HIF-1alpha and VEGF. Further studies are required to demonstrate the in vivo relevance of these observations with regard to the proposed role for adenosine as a key element in hypoxia and in tumors.     

羟基红花黄色素A上调低氧状态下血管内皮细胞中缺氧诱导因子-1α的表达

羟基红花黄色素A(hydroxysafflor yellow A，HSYA)是从菊科植物红花中提取的查尔酮类化合物。本实验观察HSYA对低氧状态(1% O₂)下人脐静脉内皮细胞株(Eahy926)增殖的促进作用及其调节Eahy 926细胞中林希氏基因(von Hippel-Lindau gene，VHL)，抑癌基因p53(tumor suppressor gene p53，p53)和降解缺氧诱导因子-1α(hypoxia-inducible factor 1α，HIF-1α)的作用。采用MTT法测定低氧状态下HSYA(100、 10和1 μmol·L^-1)对Eahy 926细胞增殖率的影响。免疫组织化学方法测定VHL与p53蛋白表达数量和定位。RT-PCR方法检测细胞中VHL、 p53和HIF-1α的转录水平。Western blotting方法检测VHL、 p53和HIF-1α的蛋白表达。与低氧模型对照组相比，HSYA(100 μmol·L^-1)促使细胞增殖率升高，HIF-1α转录和蛋白表达水平显著增强，呈时间依赖性。给药8 h后，VHL与p53的蛋白表达和转录水平均明显降低。HSYA诱导HIF-1α表达增加并提高低氧状态下细胞增殖率的作用，可能通过抑制VHL和p53两种HIF-1α的降解调节因子实现。     

Disulfiram deregulates HIF-α subunits and blunts tumor adaptation to hypoxia in hepatoma cells

Aim:

Disulfiram is an aldehyde dehydrogenase inhibitor that was used to treat alcoholism and showed anticancer activity, but its anticancer mechanism remains unclear. The aim of this study was to investigate the effects of disulfiram on the hypoxia-inducible factor (HIF)-driven tumor adaptation to hypoxia in vitro.

Methods:

Hep3B, Huh7 and HepG2 hepatoma cells were incubated under normoxic (20% O₂) or hypoxic (1% O₂) conditions for 16 h. The expression and activity of HIF-1α and HIF-2α proteins were evaluated using immunoblotting and luciferase reporter assay, respectively. Semi-quantitative RT-PCR was used to analyze HIF-mediated gene expression. Endothelial tubule formation assay was used to evaluate the anti-angiogenic effect.

Results:

Hypoxia caused marked expression of HIF-1α and HIF-1α in the 3 hepatoma cell lines, dramatically increased HIF activity and induced the expression of HIF downstream genes (EPO, CA9, VEGF-A and PDK1) in Hep3B cells. HIF-2α expression was positively correlated with the induction of hypoxic genes (CA9, VEGF-A and PDK1). Moreover, hypoxia markedly increased VEGF production and angiogenic potential of Hep3B cells. Disulfiram (0.3 to 2 μmol/L) inhibited hypoxia-induced gene expression and HIF activity in a dose-dependent manner. Disulfiram more effectively suppressed the viability of Hep3B cells under hypoxia, but it did not affect the cell cycle. Overexpression of HIF-2α in Hep3B cells reversed the inhibitory effects of disulfiram on hypoxia-induced gene expression and cell survival under hypoxia.

Conclusion:

Disulfiram deregulates the HIF-mediated hypoxic signaling pathway in hepatoma cells, which may contribute to its anticancer effect. Thus, disulfiram could be used to treat solid tumors that grow in a HIF-dependent manner.     

Q39, a novel synthetic Quinoxaline 1,4-Di-N-oxide compound with anti-cancer activity in hypoxia

Hypoxia is one of the inevitable circumstances in various tumors and results in tumor resistance to radiotherapy and chemotherapy. The present data showed that 3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline 1,4-dioxide (Q39), derived from Quinoxaline 1,4-Di-N-oxide, possessed high anti-cancer activity in hypoxia. Cytotoxicity assay demonstrated that Q39 is a potential and high efficient anti-cancer compound in all tested cell lines with IC50 values of 0.18+/-0.03-8.88+/-1.12 microM in hypoxia and 0.33+/-0.04-8.74+/-1.28 microM in normoxia . In the following work concerning the mechanism of Q39 in hypoxia, we confirmed that Q39 could cause the apoptosis of K562 cells in a time-dependent manner. By fluorescence stain assay, Q39-induced mitochondria membrane potential (Delta Psi m) loss was observed in K562 cells in hypoxia. Based on the western blotting, Q39 decreased the protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in hypoxia. The compound caused the activation of caspase-3 and subsequent cleavage of its substrate poly (ADP-ribose) polymerase (PARP) in hypoxia. Meanwhile, we found the upregulation of Bax by Q39 in K562 cells as well as the downregulation of Bcl-2. Q39 also influenced the expression of Mitogen-Activated Protein Kinase (MAPKs) and other proteins relative to mitochondria induced apoptosis. In addition, Q39-mediated apoptosis was not reversed after treatment with the JNK-specific inhibitor. In summary, the present study demonstrated Q39 was a novel compound against cancer cells in hypoxia. The mitochondrial pathway mediated by Bcl-2 protein family and MAPKs and the HIF-1 pathway might be involved in signaling Q39-induced apoptosis.     

Design of hypoxia-targeting drugs as new cancer chemotherapeutics

The tumor microenvironment is now recognized as a major factor that influences not only the response to conventional anti-cancer therapies but also helps define the potential for malignant progression and metastasis. In particular, hypoxia is now considered a fundamentally important characteristic of the tumor microenvironment. Furthermore, discovery of the hypoxia inducible factor 1alpha (HIF-1alpha) has led to a rapidly increasing understanding of the molecular mechanisms involved in tumor hypoxia. This in turn has led to the current extensive interest in the signal molecules related to tumor hypoxia as potential molecular targets for cancer therapeutics. In this paper we give an overview of recent advances in hypoxia research, including cancer treatments that target tumor hypoxia. Progress in the development of hypoxia-targeting drugs will be discussed, including antiangiogenic hypoxic cell radiosensitizers and hypoxic cytotoxins, hypoxia targeting boron carriers and p53-inhibiting bifunctional radiosensitizers. We will also review our own recent research results in these areas. For example, we have found that certain of the 2-nitroimidazole radiosensitizers and heterocycle-N-oxide hypoxic cytotoxins we developed have antiangiogenic activity and antimetastatic activity. We propose that these activities are based on the inhibition of signal transduction mediated by HIF-1alpha. The anti-tumor activities of hypoxia response are considered to be cytostatic (tumor dormancy-inducing) effects in contrast to cytotoxic DNA damaging effects. The combination of these cytostatic effects that are related to radiosensitization with the cytotoxic effects of radiation should improve the prognosis and QOL of patients receiving radiation and lead to an overall response to treatment. Based on these considerations, we developed the antiangiogenic hypoxic cell radiosensitizers, TX-1877, TX-1898 and the hypoxic cytotoxin TX-402 that inhibits the HIF-1alpha pathway We will also discuss our research involved with the development of other drugs to exploit tumor hypoxia, including a hypoxia-targeting boron carrier for boron neutron capture therapy (BNCT) and a p53 inhibiting radiosensitizer.     

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning.