leupeptins and cobaltous-chloride

Functional imaging has become an important tool in oncology because it not only provides information about the size and localization of the tumour, but also about the pathophysiological features of the tumoural cells. One of the characteristic features of some tumour types is that their fast growth leads to deficient intratumoral vascularization, which results in low oxygen availability. To overcome this lack of oxygen, tumoural cells activate the neoangiogenic program by upregulating the transcription factor HIF-1α. Herein we report a non-invasive in vitro detection method of hypoxia using designed fluorescent peptide probes based on the oxygen-dependent degradation domain of HIF-1α. The fluorescent probe retains the oxygen-sensing capability of HIF-1α, so that it is stabilized under hypoxia and readily degraded by the proteasome under normoxia, thus providing direct information of the cellular oxygen availability.

Topics: Animals; Biosensing Techniques; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cobalt; Female; Fluorescent Dyes; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Mice; Mice, Inbred BALB C; Mice, Nude; Mimosine; Oxygen; Peptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Domains; Proteolysis; Spectrometry, Fluorescence

Vascular endothelial growth factor (VEGF)-dependent angiogenesis is crucial for corpus leteum formation and their functional maintenance in mammalian ovaries. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated transcriptional activation contributes to the increased expression of VEGF gene in response to hypoxia in the bovine developing luteal cells (LCs). By real-time RT-PCR analysis, VEGF messenger RNA (mRNA) expression was found to significantly increase under hypoxia or treatment with desferrioxamine (DFX), cobalt chloride (CoCl(2)) or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG-132), while these increased VEGF mRNA expressions could also be blocked by ferrous ammonium sulfate (FAS) or cis-element oligodeoxynucleotide (dsODN) transfection under hypoxia. Further analysis also found that these changes of VEGF mRNA were consistent with HIF-1α expression or HIF-1 activity. Taken together, our results indicate that VEGF is transcriptionally activated by hypoxia through HIF-1α-mediated mechanisms in LCs. This hypoxia-induced transcriptional activation may be one of the important mechanisms mediating the increase of VEGF expression in developing LCs during mammalian corpus leteum formation.

Topics: Animals; Blotting, Western; Cattle; Cell Hypoxia; Cobalt; Deferoxamine; Female; Ferrous Compounds; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Luteal Cells; Oligodeoxyribonucleotides; Quaternary Ammonium Compounds; Real-Time Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transcriptional Activation; Vascular Endothelial Growth Factor A

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1alpha (HIF-1alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1alpha protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl(2) treatment, both well known for induction of HIF-1alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1alpha up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1alpha protein accumulation. Blockage of HIF-1alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1alpha protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1alpha-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1alpha to block HIF-1alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1alpha-dependent up-regulation of PAI-1.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cobalt; Cycloheximide; Cysteine-Rich Protein 61; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Kinetics; Leupeptins; MAP Kinase Signaling System; Neoplasm Invasiveness; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Protein Biosynthesis; Protein Synthesis Inhibitors; Recombinant Proteins; RNA, Messenger; Stomach Neoplasms; Up-Regulation

Placental hypoxia has been implicated in pregnancy pathologies, including fetal growth restriction and preeclampsia; however, the mechanism by which the trophoblast cell responds to hypoxia has not been adequately explored. Glucose transport, a process crucial to fetoplacental growth, is upregulated by hypoxia in a number of cell types. We investigated the effects of hypoxia on the regulation of trophoblast glucose transporter (GLUT) expression and activity in BeWo choriocarcinoma cells, a trophoblast cell model, and human placental villous tissue explants. GLUT1 expression in BeWo cells was upregulated by the hypoxia-inducing chemical agents desferroxamine and cobalt chloride. Reductions in oxygen tension resulted in dose-dependent increases in GLUT1 and GLUT3 expression. Exposure of cells to hypoxic conditions also resulted in an increase in transepithelial glucose transport. A role for hypoxia-inducible factor (HIF)-1 was suggested by the increase in HIF-1alpha as a result of hypoxia and by the increase in GLUT1 expression following treatment of BeWo with MG-132, a proteasomal inhibitor that increases HIF-1 levels. The function of HIF-1 was confirmed in experiments where the hypoxic upregulation of GLUT1 and GLUT3 was inhibited by antisense HIF-1alpha. In contrast to BeWo cells, hypoxia produced minimal increases in GLUT1 expression in explants; however, treatment with MG-132 did upregulate syncytial basal membrane GLUT1. Our results show that GLUTs are upregulated by hypoxia via a HIF-1-mediated pathway in trophoblast cells and suggest that the GLUT response to hypoxia in vivo will be determined not only by low oxygen tension but also by other factors that modulate HIF-1 levels.

Topics: Actins; Cell Hypoxia; Cell Line, Tumor; Choriocarcinoma; Chorionic Villi; Cobalt; Cysteine Proteinase Inhibitors; Deferoxamine; Dose-Response Relationship, Drug; Female; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 3; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Oligonucleotides, Antisense; Pregnancy; Proteasome Endopeptidase Complex; Receptors, Transferrin; Time Factors; Tissue Culture Techniques; Trophoblasts; Up-Regulation

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits and involved in the regulation of gene expression in adaptive response to hypoxia. This study reports that the inflammatory cytokine interleukin-1beta (IL-1beta) shares common features of other known HIF-1alpha-regulated genes. Both human and mouse IL-1beta genes carry multiple HIF-1-binding sites in their promoter regions and are up-regulated by hypoxia and CoCl2 in human and mouse astrocytes in parallel with up-regulation of HIF-1alpha mRNA and protein. Inhibition of HIF-1alpha degradation by proteasome inhibitor, MG-132, potentiated hypoxia-induced IL-1beta release from human astrocytes, and this response was blocked in the presence of CdCl2. Mouse astrocytes with Hif1alpha+/- genotype demonstrated attenuated up-regulation of both HIF-1alpha and IL-1beta by hypoxia and CoCl2. Mutation of HIF-1-binding sites in the IL-1beta promoter abolished hypoxia-induced transactivation of the reporter gene transfected into human astrocytes. Similarly, HIF-1 binding "decoy" oligonuleotide transfected into astrocytes inhibited both hypoxia-induced transactivation of the HIF-1 reporter gene and IL-1beta secretion from transfected astrocytes. Collectively, the evidence suggests that the transcriptional activation of IL-1beta in astrocytes exposed to hypoxia occurs via HIF-1.

Topics: Analysis of Variance; Animals; Antimutagenic Agents; Astrocytes; Blotting, Western; Cell Hypoxia; Cobalt; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Fetus; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1; Interleukin-1; Leupeptins; Mice; Mice, Transgenic; Models, Biological; Mutagenesis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection; Vascular Endothelial Growth Factor A

The present study hypothesized that superoxide (O2(-)*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O2(-)* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl(2)-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O2(-)* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22(phox), into RMICs markedly increased HIF-1alpha levels. In contrast, the OH* scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O2(-)* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O2(-)* levels induce HIF-1alpha degradation independently of H(2)O(2) and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Hypoxia; Cell Nucleus; Cell Separation; Cobalt; DNA, Complementary; Free Radical Scavengers; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Medulla; Leupeptins; Male; NADPH Oxidases; Oligodeoxyribonucleotides, Antisense; Oxidation-Reduction; Rats; Rats, Wistar; Reactive Oxygen Species; Transcription Factors; Transcriptional Activation; Vitamin K 3; Xanthine Oxidase

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1alpha subunit and a constititutively expressed HIF-1beta subunit. Under hypoxic conditions, the HIF-1alpha subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1alpha-HIF-1beta heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl(2). Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1alpha and HIF-1-dependent transcription induced by hypoxia or CoCl(2). Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl(2), while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1alpha stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1alpha fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1alpha stabilization by CoCl(2). These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.

Topics: Cell Hypoxia; Chromones; Cobalt; DNA-Binding Proteins; Enzyme Inhibitors; Glucose Transporter Type 1; Helix-Loop-Helix Motifs; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Monosaccharide Transport Proteins; Morpholines; Nuclear Proteins; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Recombinant Fusion Proteins; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured

Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.

Topics: Breast Neoplasms; Cell Division; Cell Hypoxia; Cobalt; Cysteine Endopeptidases; Endothelial Growth Factors; Estradiol; Estrogen Receptor alpha; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Signaling Peptides and Proteins; Leupeptins; Lymphokines; Multienzyme Complexes; Promoter Regions, Genetic; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteins; Receptors, Estrogen; Response Elements; Sp1 Transcription Factor; Transcription Factors; Transcriptional Activation; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The present study was designed to test the hypothesis that hypoxia-inducible factor-1alpha (HIF-1alpha)-mediated transcriptional activation contributes to increased expression of heme oxygenase (HO) genes in renal medullary interstitial cells (RMICs). By Northern blot analysis, HO-1 mRNA expression was found to significantly increase in response to reduction of PO(2) in culture medium. However, HO-2 mRNA was not altered by hypoxia. This hypoxia-induced upregulation of HO-1 mRNA was significantly blocked by HIF-1alpha inhibition with ferrous ammonium sulfate. To further determine the role of HIF-1alpha in the activation of HO-1, the inducers of HIF-1alpha were used to address whether induction of HIF-1alpha stimulates HO-1 mRNA expression. Both desferrioxamine and CoCl(2) markedly increased HIF-1alpha mRNA and protein levels and resulted in the upregulation of HO-1 mRNA but not HO-2. Furthermore, inhibition of HIF-1alpha degradation by CBZ-LLL, an inhibitor of ubiquitin-proteasome, significantly increased HIF-1alpha protein and HO-1 mRNA but not HO-2 in these cells. Using cis-element oligodeoxynucleotide transfection to specifically decoy HIF-1alpha and block HIF-1alpha binding, increased mRNA expression of HO-1 in response to hypoxia and CoCl(2) was attenuated. In vitro nuclear run-on assays further confirmed that hypoxia and alterations of HIF-1alpha mRNA or protein levels significantly affected the formation of HO-1 mRNA. Taken together, our results indicate that HO-1, but not HO-2, is transcriptionally activated by hypoxia through HIF-1alpha-mediated mechanism in RMICs. This hypoxia-induced transcriptional activation may be one of the important mechanisms mediating increased expression of HO-1 in the renal medulla.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Hypoxia; Cobalt; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes; Kidney Medulla; Leupeptins; Male; Rats; Rats, Wistar; RNA, Messenger; Transcription Factors; Transcription, Genetic; Transfection