Reactive oxygen species attenuate nitric-oxide-mediated hypoxia-inducible factor-1alpha stabilization

Tissue hypoxia/ischemia are major pathophysiological determinants. Conditions of decreased oxygen availability provoke accumulation and activation of hypoxia-inducible factor-1 (HIF-1). Recent reports demonstrate a crucial role of HIF-1 for inflammatory events. Regulation of hypoxic responses by the inflammatory mediators nitric oxide (NO) and reactive oxygen species (ROS) is believed to be of pathophysiolgical relevance. It is reported that hypoxic stabilization of HIF-1alpha can be antagonized by NO due to its ability to attenuate mitochondrial electron transport. Likely, the formation of ROS could contribute to this effect. As conflicting results emerged from several studies showing either decreased or increased ROS production during hypoxia, we used experiments mimicking hypoxic intracellular ROS changes by using the redox cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates superoxide inside cells. Treatment of A549, HEK293, HepG2, and COS cells with DMNQ resulted in a concentration-dependent raise in ROS which correlated with HIF-1alpha accumulation. By using a HIF-1alpha-von Hippel-Lindau tumor suppressor protein binding assay, we show that ROS produced by DMNQ impaired prolyl hydroxylase activity. When HIF-1alpha is stabilized by NO, low concentrations of DMNQ (<1 microM) revealed no effect, intermediate concentrations of 1 to 40 microM DMNQ attenuated HIF-1alpha accumulation and higher concentrations of DMNQ promoted HIF-1alpha stability. Attenuation of NO-induced HIF-1alpha stability regulation by ROS was mediated by an active proteasomal degradation pathway. In conclusion, we propose that scavenging of NO by ROS and vice versa attenuate HIF-1alpha accumulation in a concentration-dependent manner. This is important to fully elucidate HIF-1alpha regulation under inflammatory conditions.     

Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1alpha accumulation--implications for prolyl hydroxylase activity and iron

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)-HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1 in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1 accumulation in different tumor cell lines. Hypoxia-induced HIF-1 accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F₀F₁-ATPase, prevented hypoxia-induced HIF-1 protein accumulation and had no effect on HIF-1 induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1 content was pronounced in cells exposed to hypoxia (1.5% O₂) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1 regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1 accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function. hypoxia-inducible factor 1; oxygen sensing     

Peroxynitrite as an alternative donor of oxygen in HIF-1alpha proline hydroxylation under low oxygen availability

In the last years, nitric oxide (NO) mediated signaling became an integral component in understanding physiological and pathophysiological processes of cell proliferation, death or cellular adaptation. Among other activities, NO affects multiple targets that allow regulation of gene expression. Recently, NO was found to attenuate accumulation of hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions because of several mechanisms: redistribution of oxygen toward non-respiratory oxygen-dependent targets (like HIF-1alpha proline hydroxylases--PHDs, which perform hydroxylation of Pro402/564 of HIF-1alpha leading to its proteasomal degradation); in addition, peroxynitrite formed during interactions between NO and mitochondria derived superoxide leads to an increase in cytosolic iron/2-oxoglutarate (2-OG), which required for PHD activation. Here, we propose a hypothesis that peroxynitrite, formed in the cells upon exposure to NO under low oxygen availability, serves as an alternative donor of oxygen for activated PHDs so they can perform HIF-1alpha proline hydroxylation to de-accumulate the protein.     

Induction of autophagic cell death by a novel molecule is increased by hypoxia

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.     

HIF-1 regulation: not so easy come, easy go

The hypoxia-inducible factor-1 (HIF-1) is the master regulator of the cellular response to hypoxia and its expression levels are tightly controlled through synthesis and degradation. It is widely accepted that HIF-1alpha protein accumulation during hypoxia results from inhibition of its oxygen-dependent degradation by the von Hippel Lindau protein (pVHL) pathway. However, recent data describe new pVHL- or oxygen-independent mechanisms for HIF-1alpha degradation. Furthermore, the hypoxia-induced increase in HIF-1alpha levels is facilitated by the continued translation of HIF-1alpha during hypoxia despite the global inhibition of protein translation. Recent work has contributed to an increased understanding of the mechanisms that control the translation and degradation of HIF-1alpha under both normoxic and hypoxic conditions.