Effect of metal ions on HIF-1α and Fe homeostasis in human A549 cells

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1α protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt–glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1α stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 μM of FeSO₄) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 μM of FeSO₄) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1α stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.     

Nickel Block of a Family of Neuronal Calcium Channels: Subtype- and Subunit-Dependent Action at Multiple Sites

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (α_1A, α_1B, α_1C, and α_1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for α_1C, followed by α_1E > α_1A > α_1B channels. In contrast, the change in activation-gating was most dramatic with α_1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for α_1A channels, but increased the nickel blocking affinity for α_1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α_1A, as coexpression with β_2a or with β₄ resulted in larger current-voltage shifts than those observed in the presence of β_1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations. Received: 12 October 1995/Revised: 17 January 1996     

Up-regulation of HIF-1α is associated with neuroprotective effects of agmatine against rotenone-induced toxicity in differentiated SH-SY5Y cells

Agmatine, a metabolite generated by arginine decarboxylation, has been reported as neuromodulator and neuroactive substance. Several findings suggest that agmatine displays neuroprotective effects in several models of neurodegenerative disorders, such as Parkinson’s disease (PD). It has been hypothesized that biogenic amines may be involved in neuroprotection by scavenging oxygen radicals, thus preventing the generation of oxidative stress. Mitochondrial dysfunction, that leads to a reduction of oxygen consumption, followed by activation of prolyl hydroxylase and decrease of hypoxia-inducible factor 1 alpha (HIF-1α) levels, has been demonstrated to play a role in PD pathogenesis. Using rotenone-treated differentiated SH-SY5Y cells as the in vitro PD model, we here investigated the molecular mechanisms underlying agmatine neuroprotective effects. Our results showed that the preliminary addition of agmatine induces HIF-1α activation, and prevents the rotenone-induced production of free radical species, and the activation of apoptotic pathways by inhibiting mitochondrial membrane potential decrease and caspase 3 as well as cytochrome c increase. Notably, these effects are mediated by HIF-1α, as indicated by experiments using a HIF-1α inhibitor. The present findings suggest that the treatment with agmatine is able to counteract the neuronal cell injury evoked by mitochondrial toxins.     

Mechanisms of tumor promotion by reactive oxygen species

This review analyzes the available information concerning mechanisms of non-genotoxic action of reactive oxygen species (ROS) during tumor promotion and pathways of their generation under the influence of chemical compounds. Special attention is given to the ability of ROS to induce pseudohypoxia through inhibition of prolyl oxidase, which is an oxygen sensor in the cell. Functions of HIF-1α as a main contributor to the ROS-induced promotion are analyzed. Data suggest that an unregulated high level of HIF-1α in the cell could induce the development of tumors. Hypothetical possibilities of ROS production under the influence of different environmental pollutants, which are promoters of tumorigenesis, include functioning of cytochrome P450 during oxidation of substrates, functioning of the mitochondrial respiratory chain, and action of peroxisome proliferators.     

Heat shock protein 90 is involved in regulation of hypoxia-driven proliferation of embryonic neural stem/progenitor cells

Hypoxia may regulate the proliferation of diverse stem cells. Our previous study showed that hypoxia promoted the proliferation of embryonic neural stem/progenitor cells (NPCs) and that hypoxia inducible factor-1(HIF-1) was critical in this process. HIF-1 could be stabilized under hypoxic conditions, and heat shock protein 90 (HSP90) is an essential protein that controls the activity and stabilization of HIF-1α. In the present work, we investigate whether HSP90 is involved in proliferation of NPCs under hypoxia by regulating HIF-1α stabilization. Geldanamycin (GA), an HSP90 inhibitor, decreased the expression of HIF-1α in NPCs during hypoxia-driven proliferation and reduced the expression level of HIF-1α protein under hypoxia in a time-dependent manner. The proliferation of NPCs induced by hypoxia was inhibited after GA treatment for 24 h. Another HSP90 inhibitor, radicicol, had the same effect on NPCs as GA. Furthermore, the expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in NPCs under hypoxia was suppressed by GA. The above data indicated that HSP90 might be involved in regulation of hypoxia-driven proliferation. Both institutes have contributed equally to this work.     

HIF-1α-dependent gene expression program during the nucleic acid-triggered antiviral innate immune responses

Recent studies suggest a novel role of HIF-1α under non-hypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers HIF-1α activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of HIF-1α protein level as well as the increase in HIF-1α target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated HIF-1α knock-down. Interestingly, HIF-1α knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that HIF-1α activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response. These authors contributed equally to this work.     

Evolution and Regulation of the Downstream Gene of Hypoxia-Inducible Factor-1α in Naked Carp (Gymnocypris przewalskii) from Lake Qinghai, China

The naked carp (Gymnocypris przewalskii) is a native teleost of Lake Qinghai (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. Hypoxia-inducible factor (HIF)-1α of Gymnocypris przewalskii was cloned and a phylogenetic tree for vertebrate HIF-1α was constructed. By analysis of maximum likelihood models of codon substitutions for HIF-1α, three positive sites in the branch lineages of crucian carp, eelpout, and flounder, and a higher proportion of neutral sites in naked carp, antarctic eelpout, rainbow trout, and grayling, were detected among all teleosts. It seems that low habitat temperatures relax the purifying selection of HIF-1α in these stenothermal coldwater fish, and both cold and hypoxic lake water contributed to the evolution of the HIF-1α gene in the naked carp. Furthermore, Glut 1 mRNA, a gene downstream from HIF-1α, has a time-course- and tissue-specific dependent response to hypoxic challenge.     

Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms

Hypoxia presents pro-apoptotic and anti-apoptotic biphasic effects that appear to be dependent upon cell types and conditions around cells. The substantial reports demonstrated that commonly used hypoxia-mimetic agents cobalt chloride (CoCl₂) and desferrioxamine (DFO) could also induce apoptosis in many different kinds of cells, but the mechanism was poorly understood. In this work, we compare the apoptosis-inducing effects of these two hypoxia-mimetic agents with acute myeloid leukemic cell lines NB4 and U937 as in vitro models. The results show that both of them induce these leukemic cells to undergo apoptosis with a loss of mitochondrial transmembrane potentials (ΔΨ m), the activation of caspase-3/8 and the cleavage of anti-apoptotic protein Mcl-1, together with the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) protein, a critical regulator for the cellular response to hypoxia. Metavanadate and sodium nitroprusside significantly abrogate DFO rather than CoCl₂-induced mitochondrial Δ Ψ m collapse, caspase-3/8 activation, Mcl-1 cleavage and apoptosis, but they fail to influence DFO and CoCl₂-induced HIF-1α protein accumulation. Moreover, inducible expression of HIF-1α gene dose not alter DFO and CoCl₂-induced apoptosis in U937 cells. In conclusion, these results propose that although both DFO and CoCl₂-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1α-independent mechanisms, DFO and CoCl₂-induced apoptosis involves different initiating signal pathways that remain to be investigated.     

Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 microM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 microM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.     

Effect of hypoxia-inducible factor-1alpha silencing on the sensitivity of human brain glioma cells to doxorubicin and etoposide

Multidrug resistance (MDR) is a significant problem underlying the poor prognosis associated with gliomas. Hypoxia-inducible factor-1α (HIF-1α) is thought to induce the genes expression involved in MDR. To evaluate the effect of silencing HIF-1α in human glioma T98G cells, cells were transfected with HIF-1α-small interference RNA (HIF-1α-siRNA) and cultured under hypoxic conditions. The effect of HIF-1α-siRNA on HIF-1α and multidrug resistance-associated protein 1 gene (MRP1) and protein levels was determined. Silencing rates of HIF-1α were 90%, 85%, and 88% at 24, 48, 72 h post-transfection, respectively. Corresponding rates of HIF-1α protein were 74.5%, 61.1% and 59.1%. MRP1 protein levels decreased by 7.6%, 36.8% and 45.2%. HIF-1α-siRNA transfected cells were significantly more sensitive to doxorubicin and etoposide compared to non-transfected cells. These findings suggest that the HIF-1α plays a role in mediating chemotherapeutic drug resistance in glioma cells. HIF-1α silencing may prove to be an effective therapeutic means of treating gliomas.     

Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

The Role of Hypoxia in the Differentiation of P19 Embryonal Carcinoma Cells into Dopaminergic Neurons

Nervous system development at early stage is in hypoxic environment. Very little is known about the role of hypoxia in neuronal development. P19 embryonal carcinoma (EC) cells are a widely used model for studying early neuronal development. In this study we investigated the roles of hypoxia in differentiation of dopaminergic neurons derived from P19 EC cells. Results demonstrate that hypoxia increases the percentage of differentiated neurons, especially neurons of dopaminergic phenotype. To investigate the potential mechanism involved in hypoxia promoted differentiation of dopaminergic neurons, we measured the expression of hypoxia-inducible factor 1α (HIF-1α), based on its characteristic response to hypoxia. The result shows that HIF-1α mRNA level in P19 EC cells increases after hypoxia treatment. It is known that HIF-1α regulates the expression of tyrosine hydroxylase (TH) gene through binding to its promoter. Therefore, we propose that the underlying mechanism for hypoxia promoted differentiation of dopaminergic neurons was mediated by HIF-1α up-regulation under hypoxia. Yue Wang—Co-first author. Special Issue in honor of Dr. Ji-Sheng Han.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

Hypoxia-induced compensatory effect as related to Shh and HIF-1α in ischemia embryo rat heart

Chronic cardiac ischemia/hypoxia induces coronary collateral formation and cardiomyocyte proliferation. Hypoxia can induce cellular adaptive responses, such as synthesis of VEGF for angiogenesis and IGF-2 for proliferation. Both reduce apoptotic effects to minimize injury or damage. To investigate the mechanism of neoangiogenesis and proliferation of fetal heart under umbilical cord compression situation, we used H9c2 cardiomyoblast cell culture, and in vivo embryonic hearts as our study models. Results showed hypoxia induced not only the increase of IGF-2 and VEGF expression but also the activation of their upstream regulatory genes, HIF-1α and Shh. The relationship between HIF-1α and Shh was further studied by using cyclopamine and 2-ME2, inhibitor of Shh and HIF-1α signaling, respectively, in the cardiomyoblast cell culture under hypoxia. We found that the two inhibitors not only blocked their own signal pathway, but also inhibited each other. The observations revealed when fetal heart under hypoxia that HIF-1α and Shh pathways maybe involve in cell proliferation and neoangiogenesis to minimize injury or damage, whereas the complex cross-talk between the two pathways remains unknown. Pei-Cheng Lin, Chih-Yang Huang, and Wei-Wen Kuo contributed equally to this work.