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1.  Natural Product-Based Inhibitors of Hypoxia-Inducible Factor-1 (HIF-1) 
Current drug targets  2006;7(3):355-369.
The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates the expression of more than 70 genes involved in cellular adaptation and survival under hypoxic stress. Activation of HIF-1 is associated with numerous physiological and pathological processes that include tumorigenesis, vascular remodeling, inflammation, and hypoxia/ischemia-related tissue damage. Clinical studies suggested that HIF-1 activation correlates directly with advanced disease stages and treatment resistance among cancer patients. Preclinical studies support the inhibition of HIF-1 as a major molecular target for antitumor drug discovery. Considerable effort is underway, in government laboratories, industry and academia, to identify therapeutically useful small molecule HIF-1 inhibitors. Natural products (low molecular weight organic compounds produced by plants, microbes, and animals) continue to play a major role in modern antitumor drug discovery. Most of the compounds discovered to inhibit HIF-1 are natural products or synthetic compounds with structures that are based on natural product leads. Natural products have also served a vital role as molecular probes to elucidate the pathways that regulate HIF-1 activity. Natural products and natural product-derived compounds that inhibit HIF-1 are summarized in light of their biological source, chemical class, ancd effect on HIF-1 and HIF-mediated gene regulation. When known, the mechanism(s) of action of HIF-1 inhibitors are described. Many of the substances found to inhibit HIF-1 are non-druggable compounds that are too cytotoxic to serve as drug leads. The application of high-throughput screening methods, complementary molecular-targeted assays, and structurally diverse chemical libraries hold promise for the discovery of therapeutically useful HIF-1 inhibitors.
PMCID: PMC2908043  PMID: 16515532
HIF-1; Natural Product; Tumor Hypoxia; Molecular-Targeted Drug Discovery; Small Molecule HIF-1 Inhibitor; Hypoxia Selective
2.  STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells 
Oncogene  2013;33(13):1670-1679.
Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The ‘signal transducer and activator of transcription-3’ (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.
doi:10.1038/onc.2013.115
PMCID: PMC3868635  PMID: 23604114
cotranscriptional activation; HIF; hypoxia; STAT3; transcription
3.  Identification of small molecule compounds that inhibit the HIF-1 signaling pathway 
Molecular Cancer  2009;8:117.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1476-4598-8-117
PMCID: PMC2797767  PMID: 20003191
4.  Benzochromenones from the Marine Crinoid Comantheria rotula Inhibit Hypoxia-Inducible Factor-1 (HIF-1) in Cell-Based Reporter Assays and Differentially Suppress the Growth of Certain Tumor Cell Lines 
Journal of natural products  2007;70(9):1462-1466.
Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that promotes tumor cell adaptation and survival under hypoxic conditions. HIF-1 is currently recognized as an important molecular target for anti-cancer drug discovery. The NCI Open Repository of marine invertebrates and algae lipid extracts was evaluated using a T47D breast tumor cell-based reporter assay for HIF-1 inhibitory activity. Bioassay-guided fractionation of an active extract from a crinoid Comantheria rotula yielded seven benzo[g]chromen-4-one and benzo[h]chromen-4-one pigments (1–7). The structures of the new benzo[g]chromenone dimer 9,9'-oxybis-neocomantherin (1) and another new natural pigment 5 were deduced from spectroscopic and spectrometric data. The crinoid pigments significantly inhibited both hypoxia-induced and iron chelator-induced HIF-1 luciferase reporter activity in breast and prostate tumor cells. However, inhibition of HIF-1 in the reporter assay did not translate into a significant decrease in expression of the downstream HIF-1 target secreted vascular endothelial growth factor (VEGF). Compound 1 was found to inhibit tumor cell growth in the NCI 60-cell line panel (GI50 values 1.6 to 18.2 μM) and 6 produced a unique pattern of tumor cell growth suppression. Five cell lines from different organs were hypersensitive to 6 (GI50 values 0.29 to 0.62 μM) and three others were moderately sensitive (GI50 values 2.2 to 5.1 μM), while the GI50 values for most other cell lines ranged from 20 to 47 μM. Crinoid benzo[g]chromenones were also found to scavenge radicals in a modified DPPH assay.
doi:10.1021/np070224w
PMCID: PMC2910718  PMID: 17844994
5.  CDK1 stabilizes HIF-1α via direct phosphorylation of Ser668 to promote tumor growth 
Cell Cycle  2013;12(23):3689-3701.
Hypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intratumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1α expression represents an attractive strategy to sensitize hypoxic tumors to anticancer therapies. Here, we show that cyclin-dependent kinase 1 (CDK1) regulates the expression of HIF-1α, independent of its known regulators. Overexpression of CDK1 and/or cyclin B1 is sufficient to stabilize HIF-1α under normoxic conditions, whereas inhibition of CDK1 enhances the proteasomal degradation of HIF-1α, reducing its half-life and steady-state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1α at a previously unidentified regulatory site, Ser668. HIF-1α is stabilized under normoxic conditions during G2/M phase via CDK1-mediated phosphorylation of Ser668. A phospho-mimetic construct of HIF-1α at Ser668 (S668E) is significantly more stable under both normoxic and hypoxic conditions, resulting in enhanced transcription of HIF-1 target genes and increased tumor cell invasion and migration. Importantly, HIF-1α (S668E) displays increased tumor angiogenesis, proliferation, and tumor growth in vivo compared with wild-type HIF-1α. Thus, we have identified a novel link between CDK1 and HIF-1α that provides a potential molecular explanation for the elevated HIF-1 activity observed in primary and metastatic tumors, independent of hypoxia, and offers a molecular rationale for the clinical translation of CDK inhibitors for use in tumors with constitutively active HIF-1.
doi:10.4161/cc.26930
PMCID: PMC3903720  PMID: 24189531
CDK1; HIF-1α; angiogenesis; cell cycle; hypoxia
6.  Saururus cernuus Lignans - Potent Small Molecule Inhibitors of Hypoxia-Inducible Factor-1 
Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.
doi:10.1016/j.bbrc.2005.05.191
PMCID: PMC2905542  PMID: 15967416
Hypoxia; Breast Cancer; HIF-1 Inhibitor; Natural Product; Small Molecule; Dineolignan; Sesquineolignan; Manassantin; Saururus cernuus
7.  Disulfiram deregulates HIF-α subunits and blunts tumor adaptation to hypoxia in hepatoma cells 
Acta Pharmacologica Sinica  2013;34(9):1208-1216.
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.
doi:10.1038/aps.2013.52
PMCID: PMC4003155  PMID: 23852087
disulfiram; hepatoma; hypoxia; HIF-2; VEGF; angiogenesis
8.  Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells 
Marine Drugs  2015;13(11):6962-6976.
Hypoxia inducible factor-1α (HIF-1α) is an essential regulator of the cellular response to low oxygen concentrations, activating a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1α is overexpressed in various cancers and therefore represents a considerable chemotherapeutic target. Salternamide A (SA), a novel small molecule that is isolated from a halophilic Streptomyces sp., is a potent cytotoxic agent against a variety of human cancer cell lines. However, the mechanisms by which SA inhibits tumor growth remain to be elucidated. In the present study, we demonstrate that SA efficiently inhibits the hypoxia-induced accumulation of HIF-1α in a time- and concentration-dependent manner in various human cancer cells. In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions. Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells. Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.
doi:10.3390/md13116962
PMCID: PMC4663561  PMID: 26610526
salternamide A (SA); HIF-1α; PI3K/Akt/mTOR; p42/p44 MAPK; STAT3; cell death
9.  Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response 
Cellular signalling  2013;25(9):1895-1903.
Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactoral enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 require distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity.
doi:10.1016/j.cellsig.2013.05.018
PMCID: PMC3700616  PMID: 23707522
hypoxia; HIF; enhanceosome; transcription factors; tumor microenvironment; transcription
10.  Molecular-Targeted Antitumor Agents 19 
Journal of natural products  2008;71(11):1854-1860.
A natural product chemistry-based approach was employed to discover small molecule inhibitors of the important tumor-selective molecular target hypoxia-inducible factor-1 (HIF-1). Bioassay-guided isolation of an active lipid extract of a Saipan collection of the marine sponge Lendenfeldia sp. afforded the terpene-derived furanolipid furospongolide as the primary inhibitor of hypoxia-induced HIF-1 activation (IC50 2.9 μM, T47D breast tumor cells). The active component of the extract also contained one new cytotoxic scalarane sesterterpene and two previously reported scalaranes. Furospongolide blocked the induction of the downstream HIF-1 target secreted vascular endothelial growth factor (VEGF) and was shown to suppress HIF-1 activation by inhibiting the hypoxic induction of HIF-1α protein. Mechanistic studies indicate that furospongolide inhibits HIF-1 activity primarily by suppressing tumor cell respiration via the blockade of NADH-ubiquinone oxidoreductase (complex I)-mediated mitochondrial electron transfer.
doi:10.1021/np800342s
PMCID: PMC2893247  PMID: 18989978
11.  Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions 
Oncology Letters  2016;12(2):1034-1040.
Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic conditions in HepG2 cells via the TLR4 mediated pathway, suggesting that the HIF-1α/TLR4 signaling cohort may act as a novel therapeutic target for the treatment of hepatocellular cancer.
doi:10.3892/ol.2016.4705
PMCID: PMC4950743  PMID: 27446390
hypoxia inducible factor-1α; toll-like receptor 4 signaling pathway; HepG2 cell line; anti-tumor effects
12.  Tight Control of Hypoxia-inducible Factor-α Transient Dynamics Is Essential for Cell Survival in Hypoxia 
The Journal of Biological Chemistry  2014;289(9):5549-5564.
Background: Hypoxia inducible factor-α (HIF-α) is the main transcription factor activated in low oxygen conditions.
Results: Single cell imaging reveals pulses in nuclear levels of HIF-α.
Conclusion: The transient nature of the HIF-α nuclear accumulation is required to avoid cell death.
Significance: The duration of HIF-α response depends on cellular oxygenation, and can encode information and dictate cell fate.
Intracellular signaling involving hypoxia-inducible factor (HIF) controls the adaptive responses to hypoxia. There is a growing body of evidence demonstrating that intracellular signals encode temporal information. Thus, the dynamics of protein levels, as well as protein quantity and/or localization, impacts on cell fate. We hypothesized that such temporal encoding has a role in HIF signaling and cell fate decisions triggered by hypoxic conditions. Using live cell imaging in a controlled oxygen environment, we observed transient 3-h pulses of HIF-1α and -2α expression under continuous hypoxia. We postulated that the well described prolyl hydroxylase (PHD) oxygen sensors and HIF negative feedback regulators could be the origin of the pulsatile HIF dynamics. We used iterative mathematical modeling and experimental analysis to scrutinize which parameter of the PHD feedback could control HIF timing and we probed for the functional redundancy between the three main PHD proteins. We identified PHD2 as the main PHD responsible for HIF peak duration. We then demonstrated that this has important consequences, because the transient nature of the HIF pulse prevents cell death by avoiding transcription of p53-dependent pro-apoptotic genes. We have further shown the importance of considering HIF dynamics for coupling mathematical models by using a described HIF-p53 mathematical model. Our results indicate that the tight control of HIF transient dynamics has important functional consequences on the cross-talk with key signaling pathways controlling cell survival, which is likely to impact on HIF targeting strategies for hypoxia-associated diseases such as tumor progression and ischemia.
doi:10.1074/jbc.M113.500405
PMCID: PMC3937633  PMID: 24394419
Cell Death; Hypoxia; Hypoxia-inducible Factor; Imaging; Mathematical Modeling; Negative Feedback Loop; p53; Prolyl Hydroxylase
13.  Molecular-Targeted Antitumor Agents 15: Neolamellarins from the Marine Sponge Dendrilla nigra Inhibit Hypoxia-Inducible Factor-1 (HIF-1) Activation and Secreted Vascular Endothelial Growth Factor (VEGF) Production in Breast Tumor Cells 
Journal of natural products  2007;70(11):1741-1745.
The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) has emerged as a major antitumor molecular target. Inhibition of HIF-1 activation has been shown to suppress the growth, survival, and metastatic spread of hypoxic tumors. The NCI Open Repository of marine invertebrates and algae lipid extracts was evaluated for HIF-1 inhibitory activity in a T47D human breast tumor cell-based reporter assay. Bioassay-guided chromatographic separation of the active extract from the sponge Dendrilla nigra produced four new lamellarin-like phenolic pyrroles, which most closely resemble the structure of the known D. cactos compound lamellarin O. However, unlike lamellarins, the structures of neolamellarin A (1), neolamellarin B (2), 5-hydroxyneolamellarin B (3), and 7-hydroxyneolamellarin A (4) lack the carboxyl moiety at position C-2 of the substituted pyrrole ring and have a significantly different pattern of oxidation. Compound 4 was found to inhibit hypoxia-induced HIF-1 activation (IC50 1.9 μM) in T47D cells. Hypoxic induction of vascular endothelial growth factor (VEGF), a potent angiogenic factor and HIF-1 target gene, was also inhibited by 4 at the secreted protein level.
doi:10.1021/np070206e
PMCID: PMC2914556  PMID: 17958397
14.  The Caulerpa Pigment Caulerpin Inhibits HIF-1 Activation and Mitochondrial Respiration 
Journal of natural products  2009;72(12):2104-2109.
The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. 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. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.
doi:10.1021/np9005794
PMCID: PMC2798910  PMID: 19921787
15.  Hypoxia-inducible factor–2 (HIF-2) regulates hepatic erythropoietin in vivo 
Journal of Clinical Investigation  2007;117(4):1068-1077.
Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1α and/or Hif-2α (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel–Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1α. Here we show that conditional inactivation of Hif-2α in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2α but not Hif-1α and that the hypoxic induction of liver Epo in anemic adults was Hif-2α dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1α dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.
doi:10.1172/JCI30117
PMCID: PMC1838939  PMID: 17404621
16.  Identification of a novel small molecule HIF-1α translation inhibitor 
Purpose
Hypoxia inducible factor-1 (HIF-1) is the central mediator of the cellular response to low oxygen and functions as a transcription factor for a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1 is over-expressed in cancer and has become an important therapeutic target in solid tumors. In this study, a novel HIF-1α inhibitor was identified and its molecular mechanism was investigated.
Experimental Design
Using a HIF-responsive reporter cell-based assay, a 10,000-membered natural product-like chemical compound library was screened to identify novel HIF-1 inhibitors. This led us to discover KC7F2, a lead compound with a central structure of cystamine. The effects of KC7F2 on HIF-1 transcription, translation and protein degradation processes were analyzed.
Results
KC7F2 markedly inhibited HIF-mediated transcription in cells derived from different tumor types, including glioma, breast and prostate cancers and exhibited enhanced cytotoxicity under hypoxia. KC7F2 prevented the activation of HIF-target genes such as Carbonic Anhydrase IX, Matrix Metalloproteinase 2 (MMP2), Endothelin 1 and Enolase 1. Investigation of the mechanism of action of KC7F2 showed that it worked through the down-regulation of HIF-1α protein synthesis, an effect accompanied by the suppression of the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and p70 S6 kinase (S6K), key regulators of HIF-1α protein synthesis.
Conclusion
These results show that KC7F2 is a potent HIF-1 pathway inhibitor and that its potential as a cancer therapy agent warrants further study.
doi:10.1158/1078-0432.CCR-08-3180
PMCID: PMC2770235  PMID: 19789328
17.  A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery 
BMC Cell Biology  2008;9:18.
Background
Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.
Results
Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the β domain of VHL and a hydroxyproline-containing HIFα peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFα peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.
Conclusion
The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.
doi:10.1186/1471-2121-9-18
PMCID: PMC2346465  PMID: 18402654
18.  The Hypoxia-Associated Factor Switches Cells from HIF-1α– to HIF-2α–Dependent Signaling Promoting Stem Cell Characteristics, Aggressive Tumor Growth and Invasion 
Cancer Research  2011;71(11):4015-4027.
Most solid tumors and their metastases experience periods of low oxygen or hypoxia, which is of major clinical significance as it promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1α and HIF-2α. The HIFs are nonredundant and regulate both overlapping and unique downstream target genes. Here, we describe a novel mechanism for the switch between HIF-1α– and HIF-2α–dependent transcription during tumor hypoxia caused by the hypoxia associated factor (HAF). HAF is overexpressed in a variety of tumors and its levels are decreased during acute hypoxia, but increased following prolonged hypoxia. We have previously identified HAF as an E3 ubiquitin ligase that binds and ubiquitinates HIF-1α by an oxygen and pVHL-independent mechanism, thus targeting HIF-1α for proteasomal degradation. Here, we show that HAF also binds to HIF-2α, but at a different site than HIF-1α, and increases HIF-2α transactivation without causing its degradation. HAF, thus, switches the hypoxic response of the cancer cell from HIF-1α–dependent to HIF-2α–dependent transcription and activates genes involved in invasion such as MMP9, PAI-1, and the stem cell factor OCT-3/4. The switch to HIF-2α–dependent gene expression caused by HAF also promotes an enriched tumor stem cell population, resulting in highly aggressive tumors in vivo. Thus, HAF, by causing a switch from a HIF-1α– to HIF-2α–dependent response to hypoxia, provides a mechanism for more aggressive growth of tumors under prolonged hypoxia.
doi:10.1158/0008-5472.CAN-10-4142
PMCID: PMC3268651  PMID: 21512133
19.  AB106. Differential regulation of LncRNA-SARCC suppresses VHL-mutant RCC cell proliferation yet promotes VHL-normal RCC cell proliferation via modulating androgen receptor/HIF-2α/C-MYC axis under hypoxia 
Translational Andrology and Urology  2016;5(Suppl 1):AB106.
Objective
It is well established that hypoxia contributes to tumor progression in a HIF-2α-dependent manner in renal cell carcinoma (RCC), yet the role of LncRNAs involved in hypoxia-mediated RCC progression remains unclear. Here we demonstrate that LncRNA-SARCC is differentially regulated by hypoxia in a VHL-dependent manner both in tissue culture and in human RCC clinical samples. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells. Mechanism dissection reveals that LncRNA-SARCC can post-transcriptionally regulate androgen receptor (AR) by physically binding and destabilizing AR protein to suppress AR/HIF-2α/C-MYC signals. In return, HIF-2α can transcriptionally regulate the LncRNA-SARCC expression via binding to hypoxia responsive elements (HREs) on the promoter of LncRNA-SARCC. The negative feedback modulation between LncRNA-SARCC/AR complex and HIF-2α signaling may then lead to differentially modulate RCC progression in a VHL-dependent manner. Together, these results may provide us a new therapeutic approach via targeting this newly identified signal from LncRNA-SARCC to AR-mediated HIF-2α/C-MYC signals against RCC progression.
Methods
Human samples—surgical specimens from human ccRCC tissues were obtained from 16 patients in the Department of Urology, Shanghai Tenth People’s Hospital, Tongji Medical School (Shanghai, China), freshly frozen in liquid nitrogen and stored at −80 °C until use. OCT-embedded blocks were sectioned until cut planes were >70% tumor. Sections were collected for DNA, RNA, and protein extraction. Samples were cataloged, clinical information on cases was obtained through chart review, and patient identifiers were removed before analysis. Informed consent was obtained from patients and the study was approved by the Institutional Review Board of Tongji Medical College. Immunohistochemistry—immunohistochemical staining was performed as previously described [40], with antibodies specific for HIF-1α, HIF-2α, C-MYC, Ki-67, AR (Abcam Inc., Cambridge, MA, USA; 1:200 dilution) and CAIX (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200 dilution). The reactivity degree was assessed by at least two pathologists without knowledge of the clinicopathological features of tumors. The degree of positivity was initially classified according to the percentage of positive tumor cells as the following: (−) >5% cells positive, (1+) 6–25% cells positive, (2+) 26–50% cells positive, and (3+) >50% cells positive. For AR, HIF-1α, HIF-2α, C-MYC, Ki-67 and CAIX, samples were scored as negative/weak, intermediate, or strong. Only cells with clear tumor cell morphology were scored. Cell culture and transfection—the human VHL-mut RCC cell lines SW839 (AR positive), OSRC-2 (AR positive), A498 (AR negative), 769-P (AR negative), 786-O (AR negative), and the human VHL-wt RCC cell lines Caki-1 (AR positive), Caki-2 (AR positive), and the immortalized proximal tubule epithelial cell line from normal adult human kidney HK-2 and 293T (AR positive) were originally purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and preserved in our lab. All RCC cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) in the humidified 5% CO2 environment at 37 °C. SW839-VHL cell lines were stably transfected with VHL-wt cDNA into VHL-mut SW839 cells (ATCC). To generate LncRNA-SARCC overexpressing or LncRNA-SARCC knocked-down stable clones of SW839, SW839-VHL and OSRC-2 cells were transfected with lentiviral vectors, pWPI-LncRNA-SARCC vs. pWPI-Vec or pLKO1-sh-LncRNA-SARCC vs. pLKO1-shRNA-control, with the PAX2 packaging plasmid, and PMD2G envelope plasmid, then transfected into 293T cells for 48 hours to obtain the lentivirus soup followed by cryopreservation in −80 °C for later use. The cells were transfected using the lipofectamine 3000 (Invitrogen) reverse transfection protocol, according to the manufacturer’s instructions. Hypoxia—hypoxia (0.5% O2, 5% CO2, 94.5% N2) was achieved using an In Vivo2 hypoxic workstation (Ruskinn Technologies) or in a positive pressure chamber receiving gas from a custom-mixed tank (Airgas). Oxyrase® (Oxyrase) was used as a hypoxia mimetic at a final concentration of 100 mM while CoCl2 was used as a hypoxia mimetic at a final concentration of 200 µM. RNA immunoprecipitation (RIP)—native RIP was performed as described previously [50]. Briefly, SW839, SW839-VHL and OSRC-2 cells were lysed in RIPA lysis buffer (20 mM Tris-HCl/pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) supplemented with RNase inhibitor, protease inhibitor cocktail. RNase-free DNase (NEB) (400 U) was then added to the lysates and incubated on-ice for 30 minutes. The cell lysates were diluted in the RIPA buffer and 50 µL of the supernatant saved as input for PCR analysis. A total of 500 µL of the supernatant was incubated with 4 µg of AR antibody overnight (normal rabbit IgG as control). Protein A/G beads were pre-blocked by 15 mg/mL BSA in PBS. Then pre-blocked beads were added to the antibody-lysate mixture and incubated for another 2 hours. The RNA/antibody complex was washed four times by RIPA buffer supplemented with RNase inhibitor, protease inhibitor cocktail. The RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s protocol and subjected to RT-qPCR analysis. For UV cross-linking and RIP, cells were first subjected to formaldehyde cross-linking and then native RIP protocol was conducted. RNA-pull down assay—LncRNA-SARCC, LncRNA-SARCC (1.6 kb), LncRNA-SARCC (1.2 kb) or LncRNA-SARCC (0.8 kb) were in vitro transcribed respectively from the PCR generated T7 promoter driven DNA fragments and biotin-labeled with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One milligram of whole-cell lysates from SW839 cells were incubated with 3 µg of purified biotinylated transcripts for 1 hour at 25 °C and complexes were isolated with streptavidin agarose beads (Invitrogen). The AR protein present in the pull-down material was detected by standard immunoblot analysis. Chromatin immunoprecipitation assay (ChIP)—cells were cross-linked with 4% formaldehyde for 10 minutes followed by cell collection and sonication with a predetermined power to yield genomic DNA fragments of 300–1,000 bp long. Lysates were precleared sequentially with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and protein A-agarose. Anti-AR antibody (2.0 µg) was added to the cell lysates and incubated at 4 °C overnight. For the negative control, IgG was used in the reaction. Cell-cycle analysis—cells were plated at a density such that they would be 50% confluent on the day of analysis. Treatment (hypoxia) was then initiated over the next several days, so that all cells were in culture for the same amount of time and at similar confluency when harvested. BrdU analysis was performed following the standard protocol (Becton Dickinson) after a 20 min pulse with 10 mM BrdU. Cells were stained with Alexa 488 anti-BrdU (Invitrogen) and 0.1 M propidium iodide and analyzed in an LSR FACS machine (Becton Dickinson). For proliferation analysis with Hoechst staining, 104 cells were plated on 6-cm2 plates, with staining and counting done according to the manufacturer’s instructions (Invitrogen). Luciferase assay—cells were plated in 24-well plates and transfected with pGL3 reporter constructs using lipofectamine (Invitrogen) according to the manufacturer’s instruction. After transfection, DMEM media was added into the culture with dihydrotestosterone (DHT) with ethanol as vehicle control. pRL-TK was used as internal control. Luciferase activity was measured by Dual-Luciferase Assay (Promega) according to the manufacturer’s manual. Subcutaneous and renal capsule implantation—SW839 cells expressing pLKO1-sh-LncRNA-SARCC vs. pLKO1-shRNA-control (2×106) and SW839-VHL cells expressing pWPI-LncRNA-SARCC vs. pWPI-mock (2×106), were subcutaneously injected into one flank of 6-week-old male athymic nude mice (NCI) (n=8 mice per group). After 8–9 weeks, mice were sacrificed, and tumors were excised and weighed. Similarly, cells were injected into left renal capsule of 6-week-old male athymic nude mice (NCI) (n=8 mice per group). After 7–8 weeks, mice were sacrificed, and tumors were excised and weighed. Studies on animals were conducted with approval from the Animal Research Ethics Committee of the University of Rochester Medical Center. Statistical analysis—unless otherwise stated, all data were shown as mean ± SD. The SPSS 12.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. The χ2 analysis and Fisher exact probability analysis were applied for comparison among the expression of AR, HIF-1α, HIF-2α, C-MYC and Ki-67 and individual clinicopathological features. Difference of tumor cells was determined by t test or analysis of variance.
Results
(I) Hypoxia differentially regulates RCC cell proliferation in a VHL-dependent manner; (II) LncRNA-SARCC is differentially modulated by hypoxia in a VHL-dependent manner and is physically associated with AR; (III) LncRNA-SARCC interacts with AR and decreases AR protein stability; (IV) LncRNA-SARCC suppressed AR which alters HIF-2α/C-MYC signals under hypoxia; (V) mechanism dissection how AR modulates HIF-2α expression at the transcriptional level under hypoxia; (VI) mechanism dissection how HIF-2α modulates LncRNA-SARCC expression at the transcriptional level under hypoxia; (VII) LncRNA-SARCC differentially modulates RCC proliferation under hypoxia in the RCC in vivo mouse model.
Conclusions
LncRNA-SARCC is differentially regulated by hypoxia in a VHL-dependent manner both in tissue culture and in human RCC clinical samples. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells.
doi:10.21037/tau.2016.s106
PMCID: PMC4842606
Renal cell carcinoma; LncRNA; hypoxia; androgen receptor
20.  Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis1 
Neuro-Oncology  2005;7(2):134-153.
Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of α and β subunits. The α subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors α and β, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.
doi:10.1215/S1152851704001115
PMCID: PMC1871894  PMID: 15831232
21.  Imaging and Targeting of the Hypoxia-inducible Factor 1-active Microenvironment 
Journal of Toxicologic Pathology  2009;22(2):93-100.
Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than normal tissues. They are refractory to radiotherapy and anticancer chemotherapy. Although more than half a century has passed since it was suggested that tumour hypoxia correlates with poor treatment outcomes and contributes to recurrence of cancer, no fundamental solution to this problem has been found. Hypoxia-inducible factor-1(HIF-1) is the main transcription factor that regulates the cellular response to hypoxia. It induces various genes, whose function is strongly associated with malignant alteration of the entire tumour. The cellular changes induced by HIF-1 are extremely important therapeutic targets of cancer therapy, particularly in therapy against refractory cancers. Therefore, targeting strategies to overcome the HIF-1-active microenvironment are important for cancer therapy. To Target HIF-1-active/ hypoxic tumor cells, we developed a fusion protein drug, PTD-ODD-Procaspase-3 that selectively induces cell death in HIF-1-active/hypoxic cells. The drug consists of the following three functional domains: the protein transduction domain (PTD), which efficiently delivers the fusion protein to hypoxic tumor cells, the ODD domain, which has a VHL-mediated protein destruction motif of human HIF-1α protein and confers hypoxia-dependent stabilization to the fusion proteins, and the human procaspase-3 proenzyme responsible for the cytocidal activity of the protein drug. In vivo imaging systems capable of monitoring HIF-1 activity in transplanted human cancer cells in mice are useful in evaluating the efficiency of these drugs and in study of HIF-1-active tumor cells.
doi:10.1293/tox.22.93
PMCID: PMC3246054  PMID: 22271982
hypoxia-inducible factor 1 (HIF-1); tumour hypoxia; hypoxia responsive element (HRE); protein transduction domain (PTD); bioluminescence; in vivo imaging
22.  Bortezomib attenuates HIF-1- but not HIF-2-mediated transcriptional activation 
Oncology Letters  2015;10(4):2192-2196.
Bortezomib is the first proteasomal inhibitor (PI) to be used therapeutically for treating relapse cases of multiple myeloma and mantle cell lymphoma. A proposed mechanism for its action is that it prevents the proteasomal degradation of proapoptotic proteins, leading to enhanced apoptosis. Although the α subunit of hypoxia-inducible factor (HIF)-1 is not degraded with bortezomib treatment, the heterodimeric HIF-1 fails to transactivate target genes. HIF-1 and HIF-2 are related hypoxia-inducible transcription factors that are important for the survival of hypoxic tumor cells. The majority of reports have focused on the effects of bortezomib on the transcriptional activities of HIF-1, but not HIF-2. The present study investigated the effects of bortezomib on HIF-2 activity in cancer cells with different levels of HIF-1α and HIF-2α subunits. HIF-α subunit levels were detected using specific antibodies, while HIF transcriptional activities were evaluated using immunodetection, reverse transcription-polymerase chain reaction and luciferase reporter assay. Bortezomib treatment was found to suppress the transcription and expression of CA9, a HIF-1-specific target gene; however, it had minimal effects on EPO and GLUT-1, which are target genes of both HIF-1 and HIF-2. These data suggest that bortezomib attenuates the transcriptional activity only of HIF-1, and not HIF-2. This novel finding on the lack of an inhibitory effect of bortezomib on HIF-2 transcriptional activity has implications for the improvement of design and treatment modalities of bortezomib and other PI drugs.
doi:10.3892/ol.2015.3545
PMCID: PMC4579903  PMID: 26622817
bortezomib; hypoxia-inducible factor; HIF-1; HIF-2; transcriptional activity
23.  Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2 
Journal of natural products  2011;74(9):1894-1901.
Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods.
doi:10.1021/np200370z
PMCID: PMC3179826  PMID: 21875114
24.  HIF-1α: a Valid Therapeutic Target for Tumor Therapy 
Hypoxia plays a major role in the induction of angiogenesis during tumor development. One mechanism by which tumor cells respond to a reduced oxygen level is via the activation of hypoxia-inducible factor-1 (HIF-1). HIF-1 is an oxygen-dependent transcriptional activator that plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1β subunit and the highly regulated HIF-1α subunits. The stability and activity of HIF-1α are regulated by various post-translational modifications, hydroxylation, acetylation, phosphorylation and sumoyaltion. Therefore, HIF-1α interacts with several protein factors including PHD, pVHL, ARD-1, SUMO and p300/CBP. Under normoxia, the HIF-1α subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)-mediated ubiquitin/proteasome pathway. The association of pVHL and HIF-1α under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, under the hypoxia condition, the HIF-1α subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Under hypoxic conditions, HIF-1 eventually acts as a master regulator of numerous hypoxia-inducible genes. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation and survival, and to glucose and iron metabolism. Moreover, it was reported that the activation of HIF-1α is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1α itself or the blocking of HIF-1α interacting proteins inhibits tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. Therefore, this review summarizes the molecular mechanism of HIF-1α stability, the biological functions of HIF-1 and its potential applications for cancer therapies.
doi:10.4143/crt.2004.36.6.343
PMCID: PMC2843877  PMID: 20368827
ARD1; Angiogenesis; Anticancer therapy; Cell proliferation/survival; Glucose metabolism; HIF-1; Iron metabolism; PHD; SUMO; pVHL; p300/CBP; Transcription factor
25.  Natural Product-Derived Small Molecule Activators of Hypoxia-Inducible Factor-1 (HIF-1) 
Current pharmaceutical design  2006;12(21):2673-2688.
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.
PMCID: PMC2907550  PMID: 16842166
HIF-1; Natural Product; Tissue Ischemia; Therapeutic Angiogenesis; Molecular-Target; Small Molecule Activator; Chemoprevention; Ischemia/Reperfusion Injury

Results 1-25 (1975560)