The von Hippel-Lindau tumor suppressor pVHL regulates the stability of Hypoxia-Inducible Factors (HIF) -1 and –2, oxygen-sensitive basic helix-loop-helix transcription factors, which mediate the hypoxic induction of angiogenic growth factors such as vascular endothelial growth factor (VEGF). Loss of VHL function results in constitutive activation of HIF-1 and HIF-2 and is associated with the development of highly vascularized tumors in multiple organs. We have used a conditional gene targeting approach to investigate the relative contributions of HIF-1 and HIF-2 to VHL-associated vascular tumorigenesis in a mouse model of liver hemangiomas. Here we demonstrate genetically that conditional inactivation of HIF-2α suppressed the development of VHL-associated liver hemangiomas and that angiogenic gene expression in hepatocytes is predominantly regulated by HIF-2 and not by HIF-1. These findings suggest that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors and that pharmacologic targeting of HIF-2 may be an effective strategy for their treatment.
Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS.
Mammalian cells have the ability to sense low oxygen levels (hypoxia). An adaptive response to hypoxia involves the induction of the transcription factor hypoxia-inducible factor 1 (HIF-1). The intracellular signaling pathways that regulate HIF-1 activation during hypoxia remain unknown. Here, we demonstrate that p38α−/− cells fail to activate HIF-1 under hypoxic conditions. Cells deficient in Mkk3 and Mkk6, the upstream regulators of p38α, also fail to activate HIF-1 under hypoxic conditions. The p38α−/− cells are able to activate HIF-1 in response to anoxia or iron chelators during normoxia. Furthermore, the hypoxic activation of p38α and HIF-1 was abolished by myxothiazol, a mitochondrial complex III inhibitor, and glutathione peroxidase 1 (GPX1), a scavenger of hydrogen peroxide. Thus, the activation of p38α and HIF-1 is dependent on the generation of mitochondrial reactive oxygen species. These results provide genetic evidence that p38 mitogen-activated protein kinase signaling is essential for HIF-1 activation.
c-Myc is frequently overexpressed in tumors and plays an important role in the regulation of cancer metabolism. Hypoxia-inducible factor-1 (HIF1), the master regulator of the hypoxic response, enhances tumorigenesis and influences metabolism via upregulation of the glycolytic pathway and suppression of mitochondrial respiration. Together, deregulated Myc and HIF1 cooperate to lend metabolic advantages to proliferating cancer cells and contribute to the Warburg Effect. Here we show that overexpression of Myc significantly stabilizes the alpha subunit of HIF1 (HIF1alpha) under normoxic conditions and enhances HIF1alpha accumulation under hypoxic conditions in cells. Post-transcriptional regulation of HIF1α by Myc led to the induction of HIF1α gene targets. Normoxic HIF1α protein expression was also dependent on Myc. Functionally; HIF1α expression was required for Myc-induced anchorage-independent growth and cell proliferation. Myc-dependent stabilization of HIF1α involved either disruption of binding to the VHL complex or post-translational protein modifications. Taken together, our findings uncover a previously uncharacterized regulatory relationship between Myc and HIF1 that has important implications for cancer metabolism and development.
Hypoxia‐inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.
ARPE‐19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme‐linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real‐time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.
HIF‐1α was readily produced by ARPE‐19 cells under hypoxia, but HIF‐2α and HIF‐3α could not be detected even after HIF‐1α silencing. HIF‐1α protein levels showed an increasing trend for the first 24 h while HIF‐1α mRNA levels fluctuated during this time. After 36 h HIF‐1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF‐1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.
HIF‐1α appears to be the main isoform of HIF functioning in ARPE‐19 cells. Under hypoxia, HIF‐1α levels are likely self‐regulated by a feedback loop that involves both transcriptional and post‐translational mechanisms. VEGF production by human RPE cells is regulated by HIF‐1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.
diabetic retinopathy; VEGF; erythropoietin; hypoxia‐inducible factor; proline hydroxylase
The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).
Patients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.
The Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.
The lipid extract of the marine sponge Mycale sp. inhibited the activation of hypoxiainducible factor-1 (HIF-1) in a human breast tumor T47D cell-based reporter assay. Bioassay-guided isolation and structure elucidation yielded 18 new lipophilic 2,5-disubstituted pyrroles, and eight structurally related known compounds. The active compounds inhibited hypoxia-induced HIF activation with moderate potency (IC50 values < 10 μM). Mechanistic studies revealed that the active compounds suppressed mitochondrial respiration by blocking NADH-ubiquinone oxidoreductase (complex I) at concentrations that inhibited HIF-1 activation. Under hypoxic conditions, reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. By inhibiting electron transport (or delivery) to complex III under hypoxic conditions, lipophilic Mycale pyrroles appear to disrupt mitochondrial ROS-regulated HIF-1 signaling.
Deregulated c-Myc occurs in ~30% of human cancers. Similarly, hypoxia-inducible factor (HIF) is commonly overexpressed in a variety of human malignancies. Under physiologic conditions, HIF inhibits c-Myc activity; however, when deregulated oncogenic c-Myc collaborates with HIF in inducing the expression of VEGF, PDK1 and hexokinase 2. Most of the knowledge of HIF derives from studies investigating a role of HIF under hypoxic conditions, however, HIF-1α stabilization is also found in normoxic conditions. Specifically, under hypoxic conditions HIF-1-mediated regulation of oncogenic c-Myc plays a pivotal role in conferring metabolic advantages to tumor cells as well as adaptation to the tumorigenic micromilieu. In addition, our own results show that under normoxic conditions oncogenic c-Myc is required for constitutive high HIF-1 protein levels and activity in Multiple Myeloma (MM) cells, thereby influencing VEGF secretion and angiogenic activity within the bone marrow microenvironment. Further studies are needed to delineate the functional relevance of HIF, MYC, and the HIF-MYC collaboration in MM and other malignancies, also integrating the tumor microenvironment and the cellular context. Importantly, early studies already demonstrate promising preclinical of novel agents, predominantly small molecules, which target c-Myc, HIF or both.
HIF; c-Myc; targeted therapy; tumor microenvironment
Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1α and constitutively expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1α inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1α activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.
cobalt sulfate heptahydrate; 7-diethylamino-4-methylcoumarin; 7,12-dimethylbenz(a)anthracence; HIF-1α; inducers; iodochlorohydroxyquinoline; NTP 1408 compound library; o-phenanthroline; qHTS
When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.
Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1α and HIF-2α proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2α interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1α and HIF-2α protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.
HIF-2α; EPO; SOD2; Hypoxia; Cobalt; USF2
Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.
Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia-inducible factor-1α (HIF-1α) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF-1α by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF-1α and Runx2 interact to activate angiogenic signals. Runx2 over-expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF-1α protein. In all cases, the Runx2 response was inhibited by siRNA-mediated suppression of HIF-1α and completely blocked by the HIF-1α inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF-1α response since VEGF is induced by hypoxia even in Runx2-null cells. Endogenous Runx2 and HIF-1α were colocalized to the nuclei of MC3T3-E1 preosteoblast cells. Moreover, HIF-1α and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF-1α on the VEGF promoter. In addition, Runx2 stimulated HIF-1α-dependent activation of an HRE-luciferase reporter gene without requiring a separate Runx2-binding enhancer. These studies indicate that Runx2 functions together with HIF-1α to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization.
Osteoblast; vascularization; angiogenesis; transcriptional factors; hypoxia
The key transcription factor that regulates the cellular responses to hypoxia is hypoxia inducible factor-1 (HIF-1). The signaling mechanisms that regulate the hypoxic activation of HIF-1 are not fully understood. Our objective here was to test whether AMP-activated kinase (AMPK) was an upstream regulator of HIF-1. Our results show that AMPK is not required for the hypoxic activation of HIF-1. Interestingly, the AMPK inhibitor, Compound C, inhibits the hypoxic activation of HIF-1 independent of AMPK. Furthermore, we demonstrate that Compound C functions as a repressor of HIF-1 by inhibiting respiration and suppressing mitochondrial generated ROS.
Hypoxia inducible factor 1; AMP-activated kinase; Compound C; hypoxia; mitochondria; reactive oxygen species
The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity
HIF asparaginyl hydroxylase (FIH) is shown to be strikingly more sensitive to peroxide than the HIF prolyl hydroxylases, indicating that hypoxia and oxidative stress are distinct regulators of the HIF response.
Hypoxic and oxidant stresses can coexist in biological systems, and oxidant stress has been proposed to activate hypoxia pathways through the inactivation of the ‘oxygen-sensing' hypoxia-inducible factor (HIF) prolyl and asparaginyl hydroxylases. Here, we show that despite reduced sensitivity to cellular hypoxia, the HIF asparaginyl hydroxylase—known as FIH, factor inhibiting HIF—is strikingly more sensitive to peroxide than the HIF prolyl hydroxylases. These contrasting sensitivities indicate that oxidant stress is unlikely to signal hypoxia directly to the HIF system, but that hypoxia and oxidant stress can interact functionally as distinct regulators of HIF transcriptional output.
FIH; HIF; hydroxylation; peroxide
Products that contain twig extracts of pawpaw (Asimina triloba, Annonaceae) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.02 μg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1α protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
The cytotoxic marine red algal metabolite thyrsiferol (1) was found to inhibit hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast tumor cells (66% inhibition at 3 μM). Compound 1 also suppressed hypoxic induction of HIF-1 target genes (VEGF, GLUT-1) at the mRNA level, and displayed tumor cell line-selective time-dependent inhibition of cell viability/proliferation. Mechanistic studies revealed that 1 selectively suppressed mitochondrial respiration at Complex I (IC50 3 μM). Thyrsiferol represents a prototypical, structurally unique electron transport chain inhibitor. The apparent rotenone-like activity may contribute to the observed cytotoxicity of 1 and play an important role in Laurencia chemical defense.
Thyrsiferol; Hypoxia-inducible factor-1 (HIF-1); Laurencia, Triterpene polyether; Marine natural product; Mitochondrial complex I inhibitor; NADH-ubiquinone oxidoreductase
Hypoxia-Inducible Factor (HIF)-1 is a dimeric protein complex that plays an integral role in the body's response to low oxygen concentrations, or hypoxia. HIF-1 is among the primary genes involved in the homeostatic process, which can increase vascularization in hypoxic areas such as localized ischemia and tumors. It is a transcription factor for dozens of target genes; HIF-1 is also essential for immunological responses and is a crucial physiological regulator of homeostasis, vascularization, and anaerobic metabolism. Furthermore, HIF-1 is increasingly studied because of its perceived therapeutic potential. As it causes angiogenesis, enhancement of this gene within ischemic patients could promote the vessel proliferation needed for oxygenation. In contrast, as HIF-1 allows for survival and proliferation of cancerous cells due to its angiogenic properties, inhibition potentially could prevent the spread of cancer. With a growing understanding of the HIF-1 pathway, the inhibition and stimulation of its transcriptional activity via small molecules is now an attractive goal. Gene therapy to achieve both vessel proliferation and tumor regression has been demonstrated in animal studies but requires significant improvement and modification before becoming commercially available. This review focuses on the potential of the HIF-1 pathway in therapeutic intervention for the treatment of diseases such as cancer and ischemia.
Hypoxia-inducible factor-1 (HIF-1) is a key mediator of oxygen homeostasis that was first identified as a transcription factor that is induced and activated by decreased oxygen tension. Upon activation, HIF-1 upregulates the transcription of genes that promote adaptation and survival under hypoxic conditions. HIF-1 is a heterodimer composed of an oxygen-regulated subunit known as HIF-1α and a constitutively expressed HIF-1β subunit. In general, the availability and activity of the HIF-1α subunit determines the activity of HIF-1. Subsequent studies have revealed that HIF-1 is also activated by environmental and physiological stimuli that range from iron chelators to hormones. Preclinical studies suggest that HIF-1 activation may be a valuable therapeutic approach to treat tissue ischemia and other ischemia/hypoxia-related disorders.
The focus of this review is natural product-derived small molecule HIF-1 activators. Natural products, relatively low molecular weight organic compounds produced by plants, animals, and microbes, have been and continue to be a major source of new drugs and molecular probes. The majority of known natural product-derived HIF-1 activators were discovered through pharmacological evaluation of specifically selected individual compounds. The combination of natural products chemistry with appropriate high-throughput screening bioassays could provide an alternative approach to discover novel natural product-derived HIF-1 activators. Potent natural product-derived HIF-1 activators that exhibit a low level of toxicity and side effects hold promise as new treatment options for diseases such as myocardial and peripheral ischemia, and as chemopreventative agents that could be used to reduce the level of ischemia/reperfusion injury following heart attack and stroke.
HIF-1; Natural Product; Tissue Ischemia; Therapeutic Angiogenesis; Molecular-Target; Small Molecule Activator; Chemoprevention; Ischemia/Reperfusion Injury
Hypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1α and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7.
Peripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO2, and 94% N2) for 24 h, and HIF-1α and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting.
HIF-1α was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1α under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1α protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1α protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1α activity in U937 cells, and HIF-1α nuclear translocation and HIF-1α binding to the VEGF promoter in A549 cells.
HDAC7 reduction in COPD causes a defect of HIF-1α induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD.
Hypoxia-inducible factor 1α (HIF1α) is an important cellular survival protein under hypoxic conditions, regulating the cellular response to low oxygen tension via recruitment of a transcriptional co-activator, p300/CBP. p300/CBP induces expression of multiple genes involved in cell survival, proliferation, angiogenesis, and tumor development. Thus, a strategy to inhibit hypoxic responses in tumors may be to target the protein-protein interaction between HIF1α and p300/CBP. Here, we document, for the first time, that the aminocoumarin antibiotic, novobiocin, directly blocks the protein-protein interaction between the HIF1α C-terminal activation domain (CTAD) and the cysteine-histidine rich (CH1) region of p300/CBP. Also, novobiocin down-regulated HIF1α-controlled gene expression, specifically CA9, which is related to tumorigenesis. In a monolayer cell culture, novobiocin inhibited cell proliferation and colony formation in the MCF-7 human breast adenocarcinoma cell line and the A549 human lung cancer cell line. Rescue experiments revealed that the recombinant CTAD fragment of HIF1α partially reversed novobiocin’s inhibitory effects on cell proliferation and colony formation in MCF-7 cells. These findings suggest a novel mechanism of action for novobiocin which has the potential for innovative therapeutic use in tumor treatment.
The transcription factors hypoxia inducible factor 1 and 2 (HIF-1 and HIF-2) regulate multiple responses to physiological hypoxia such as transcription of the hormone erythropoietin (EPO) to enhance red blood cell proliferation, vascular endothelial growth factor (VEGF) to promote angiogenesis and glycolytic enzymes to increase glycolysis. Recent studies indicate that HIFs also regulate mitochondrial respiration and mitochondrial oxidative stress. Interestingly, mitochondrial metabolism, respiration and oxidative stress also regulate activation of HIFs. In this review we examine the evidence that mitochondria and HIFs are intimately connected to regulate each other resulting in appropriate responses to hypoxia.
mitochondria; HIF; ROS; respiration
The antinociceptive and anti-inflammatory activity of caulerpin was investigated. This bisindole alkaloid was isolated from the lipoid extract of Caulerpa racemosa and its structure was identified by spectroscopic methods, including IR and NMR techniques. The pharmacological assays used were the writhing and the hot plate tests, the formalin-induced pain, the capsaicin-induced ear edema and the carrageenan-induced peritonitis. Caulerpin was given orally at a concentration of 100 μmol/kg. In the abdominal constriction test caulerpin showed reduction in the acetic acid-induced nociception at 0.0945 μmol (0.0103–1.0984) and for dypirone it was 0.0426 μmol (0.0092–0.1972). In the hot plate test in vivo the inhibition of nociception by caulerpin (100 μmol/kg, p.o.) was also favorable. This result suggests that this compound exhibits a central activity, without changing the motor activity (seen in the rotarod test). Caulerpin (100 μmol/kg, p.o.) reduced the formalin effects in both phases by 35.4% and 45.6%, respectively. The possible anti-inflammatory activity observed in the second phase in the formalin test of caulerpin (100 μmol/kg, p.o.) was confirmed on the capsaicin-induced ear edema model, where an inhibition of 55.8% was presented. Indeed, it was also observed in the carrageenan-induced peritonitis that caulerpin (100 μmol/kg, p.o.) exhibited anti-inflammatory activity, reducing significantly the number of recruit cells by 48.3%. Pharmacological studies are continuing in order to characterize the mechanism(s) responsible for the antinociceptive and anti-inflammatory actions and also to identify other active principles present in Caulerpa racemosa.
Caulerpa racemosa; antinociceptive; anti-inflammatory; caulerpin