Structure-activity relationships study of neolamellarin A and its analogues as hypoxia inducible factor-1 (HIF-1) inhibitors

The novel marine pyrrole alkaloid neolamellarin A derived from sponge has been shown to inhibit hypoxia-induced HIF-1 activity. In this work, we designed and synthesized neolamellarin A and its series of derivatives by a convergent synthetic strategy. The HIF-1 inhibitory activity and cytotoxicity of these compounds were evaluated in Hela cells by dual-luciferase reporter gene assay and MTT assay, respectively. The results showed that neolamellarin A 1 (IC₅₀ = 10.8 ± 1.0 μM) and derivative 2b (IC₅₀ = 11.9 ± 3.6 μM) had the best HIF-1 inhibitory activity and low cytotoxicity. Our SAR research focused on the effects of key regions aliphatic carbon chain length, aromatic ring substituents and C-7 substituent on biological activity, providing a basis for the subsequent research on the development of novel pyrrole alkaloids as HIF-1 inhibitors and design of small molecule probes for target protein identification.     

Suppression of hypoxia inducible factor-1alpha (HIF-1alpha) by YC-1 is dependent on murine double minute 2 (Mdm2)

Inhibition of HIF-1alpha activity provides an important strategy for the treatment of cancer. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) has been identified as an anti-HIF-1alpha drug in cancer therapy with unclear molecular mechanism. In the present study, we aimed to investigate the effect and mechanism of YC-1 on HIF-1alpha in a hepatocellular carcinoma cell line under hypoxic condition, which was generated by incubating cells with 0.1% O(2). The phenotypic and molecular changes of cells were determined by cell proliferation assay, apoptosis assay, luciferase promoter assay, and Western blot analysis. YC-1 arrested tumor cell growth in a dose-dependent manner, whereas it did not induce cell apoptosis. Hypoxia-induced upregulation of HIF-1alpha was suppressed by YC-1 administration. YC-1 inhibited HIF-1alpha protein synthesis under normoxia and affected protein stability under hypoxia. YC-1 suppressed the expression of total and phosphorylated forms of murine double minute 2 (Mdm2), whereas this inhibitory effect was blocked by overexpression of Mdm2. In conclusion, YC-1 suppressed both protein synthesis and stability of HIF-1alpha in HCC cells, and its inhibitory effects on HIF-1alpha were dependent on Mdm2.     

Cloning,characterization, hypoxia and heat shock response of hypoxia inducible factor-1 (HIF-1) from the small abalone Haliotis diversicolor

In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2833 bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919 bp encoding a protein of 590aa. Similar to other species' HIF-1, HdHIF-1 has one basic helix–loop–helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0 mg/L DO at 25 °C) stress, HdHIF-1α expression was up-regulated in gills at 4 h, 24 h and 96 h, and in hemocytes at 24 h and 96 h, while HdHIF-1β remained relatively constant. Under thermal stress (31 °C), HdHIF-1α expression was significantly increased in gills at 4 h, and hemocytes at 0 h and 4 h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor.     

Endocannabinoids participate in placental apoptosis induced by hypoxia inducible factor-1

During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed towards the relevance of endocannabinoids (ECs) and hypoxia as modulators of trophoblast cell death. However, the relation between these factors is still unknown. In this report, we evaluated the participation of ECs in placental apoptosis induced by cobalt chloride (CoCl₂), a hypoxia mimicking agent that stabilizes the expression of hypoxia inducible factor-1 alpha (HIF-1α). We found that HIF-1α stabilization decreased FAAH mRNA and protein levels, suggesting an increase in ECs tone. Additionally, CoCl₂ incubation and Met-AEA treatment reduced cell viability and increased TUNEL-positive staining in syncytiotrophoblast layer. Immunohistochemical analysis demonstrated Bax and Bcl-2 protein expression in the cytoplasm of syncytiotrophoblast. Finally, HIF-1α stabilization produced an increase in Bax/Bcl-2 ratio, activation of caspase 3 and PARP cleavage. All these changes in apoptotic parameters were reversed with AM251, a CB1 antagonist. These results demonstrate that HIF-1α may induce apoptosis in human placenta via intrinsic pathway by a mechanism that involves activation of CB1 receptor suggesting a role of the ECs in this process.     

Hypoxia-inducible factor-1 (HIF-1) promotes LDL and VLDL uptake through inducing VLDLR under hypoxia

Metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that HIF-1 (hypoxia-inducible factor-1) plays a central role in regulating glucose metabolism under hypoxia; however, the role of HIF-1 in lipid metabolism has not yet been well addressed. In the present study we demonstrate that HIF-1 promotes LDL (low-density lipoprotein) and VLDL (very-LDL) uptake through regulation of VLDLR (VLDL receptor) gene expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO (deferoxamine mesylate salt) treatment in MCF7, HepG2 and HeLa cells. Using dual-luciferase reporter and ChIP (chromatin immunoprecipitation) assays we confirmed a functional HRE (hypoxia-response element) which is localized at +405 in exon 1 of the VLDLR gene. Knockdown of HIF1A (the α subunit of HIF-1) and VLDLR, but not HIF2A (the α subunit of HIF-2), attenuated hypoxia-induced lipid accumulation through affecting LDL and VLDL uptake. Additionally we also observed a correlation between HIF-1 activity and VLDLR expression in hepatocellular carcinoma specimens. The results of the present study suggest that HIF-1-mediated VLDLR induction influences intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia.     

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0°C and after long-term warming to 5°C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0°C for the polar and 6°C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.     

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.     

Expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in pituitary tumours

The present study was performed to investigate HIF-1alpha (hypoxia-inducible factor-1alpha) expression in a large number of immunohistochemically and ultrastructurally characterized surgically removed pituitary tumours. The potential relation of HIF-1alpha with outcome variables as well as the presence of HIF-1alpha expression in the tumours treated with dopamine agonists and octreotide, a long-acting somatostatin analogue was also investigated. HIF-1alpha immunoreactivity was confined to the nucleoplasm whereas the nucleoli were unconspicuous. The distribution of HIF-1alpha was evident in the tumours whereas normal adenohypophysial cells showed no HIF-1alpha staining. HIF-1alpha expression was detected not only in the tumour cells but also in endothelial cells lining the blood vessels within the tumour. ACTH producing adenomas showed the lowest level of HIF-1alpha expression whereas pituitary carcinomas and GH producing adenomas had the highest counts. The statistical study demonstrated no significant correlation between HIF-1alpha expression, patient age, gender, tumour, size, invasiveness, cell proliferation rate and vascularity. These results suggest that the behaviour of pituitary tumours does not primarily depend of HIF-1alpha expression. Our study demonstrated an increase HIF-1alpha expression in bromocriptine treated PRL producing pituitary adenomas compared with untreated tumours but no increase in octreotide treated tumours.     

Microwave expedited synthesis of 5-aminocamptothecin analogs: Inhibitors of hypoxia inducible factor HIF-1alpha

A series of 5-aminosubstituted camptothecin analogs were prepared from the corresponding 5-hydroxycamptothecin using microwave irradiation. The analogs were assayed for ability to inhibit the action of hypoxia inducible factors (HIF-1alpha and HIF-2alpha). The 5-fluoroethyl analog showed potent inhibitory activity and is now the focus of ongoing pathway analysis and potential as an antiproliferative agent.     

Hepatocyte growth factor signaling regulates transactivation of genes belonging to the plasminogen activation system via hypoxia inducible factor-1

Hepatocyte growth factor (HGF) plays an important role in tumor growth and progression also by regulating invasive/metastatic phenotype and angiogenesis. Here we report that a molecular mechanism possibly contributing to these functions of HGF may be hypoxia inducible factor-1 (HIF-1)-dependent expression of genes of the plasminogen activation system. The following findings support this conclusion: (1) HGF enhanced the activity of a luciferase reporter construct under the control of multiple HIF-1 responsive elements (HRE) in HepG2 cells, and the cotransfection of the dominant negative for the beta-subunit (ARNT) prevented this increase; (2) HGF activated uPA and PAI-1 promoters through HIF-1 activity regulated by PI3K/JNK1 transducers, as demonstrated by cotransfection with the reporter gene promoters and the dominant negative for ARNT, p85 subunit of PI3K or JNK1; (3) hypoxia was additive to HGF in increasing reporter vector activities, but probably through different transduction pathways; (4) JNK1 wild-type expression vector increased HIF-1alpha protein expression probably in a phosphorylated state and, thus, functional for transactivating activity; and (5) c-Jun did not seem to be involved in the activation of the luciferase construct containing multiple HREs because it was not prevented by expression of TAM-67, which is the dominant negative mutant form for c-Jun.