HIF-1α Binding Research Articles

Hypoxia‐inducible factor‐1α mediates up‐regulation of neprilysin by histone deacetylase‐1 under hypoxia condition in neuroblastoma cells

Hypoxia-inducible factor (HIF)-1 is the key transcriptional activator mediating both adaptive and pathological responses to hypoxia. The purpose of this study was to find the role of HIF-1 in regulating neprilysin (NEP) at the early stage of hypoxia and explore the underlying mechanism. In this study, we demonstrated that both NEP mRNA and protein levels in neuroblastoma cells were elevated in early stages of hypoxia. Over-expression of HIF-1α gene increased NEP mRNA/protein levels, as well as enzyme activity while knockdown of HIF-1α decreased them. Meanwhile, HIF-1α was shown to bind to histone deacetylase (HDAC)-1 and reduced the association of HDAC-1 with NEP promoter, thus activating NEP gene transcription in a de-repression way. In summary, our results indicated that hypoxia in the early stages would up-regulate NEP expression, in which interaction of HIF-1α and HDAC-1 may play a role. This study suggested that NEP up-regulation might be an adaptive response to hypoxia, which was mediated by HIF-1α binding to HDAC-1 at the early stage of hypoxia.

Hyperthermia-Conditioned OECs Serum-Free–Conditioned Medium Induce NSC Differentiation Into Neuron More Efficiently by the Upregulation of HIF-1 Alpha and Binding Activity

Recent studies provide solid evidence for the importance to delineate the combined transplantation of neural stem cells (NSCs) and olfactory ensheathing cells (OECs) for the repair of central nervous system injury. One of the limitations of this approach is that the proportion of neurons differentiated from NSCs still remains at low level. Thus, how to induce NSCs to differentiate into neurons more efficiently by OECs is an attractive problem, which needs attention to be resolved. In the present study, we investigated the effects of hyperthermia-conditioned OEC-conditioned medium (OCM) on induction of NSCs into nerve cells. The conditioned medium, named 37OCM, 40OCM, and 40OCM+R (treated with HIF-1a inhibitor), were collected after OECs had been incubated at 37°C or 40°C for 6 hr, followed by incubation at 37°C for 42 hr; then, these conditioned medium were collected and used for NSC induction. Results of the present study demonstrated for the first time that 6 hr of hyperthermia (40°C) could induce OECs upregulation and the expression of hypoxia-inducible factor-1 alpha (HIF-1α). Moreover, compared with 37OCM and 40OCM+R, 40OCM could induce NSCs differentiation into neurons more efficiently, and this phenomenon is associated with the upregulation of HIF-1α after hyperthermia conditioning. The data implied that the secretory protein in OECs-cultured medium and upregulation of HIF-1α expression and binding activity induced by hyperthermia-conditioned OECs have synergistic effect to induce NSCs differentiation into a neural lineage.

HIF‐1 Alpha‐Induced Up‐Regulation of miR‐9 Contributes to Phenotypic Modulation in Pulmonary Artery Smooth Muscle Cells During Hypoxia

Pulmonary artery smooth muscle cells (PASMCs) are associated with the development of hypoxic pulmonary hypertension (HPH). Recent studies have implicated a critical role for microRNAs (miRNAs) in HPH; however, their expression and regulation in hypoxia-mediated phenotypic modulation of PASMCs remains largely unclear. Here, we report that miR-9 was induced in hypoxia and involved in a hypoxia-induced phenotypic switch in rat primary PASMCs. Knockdown of miR-9 followed by hypoxia exposure attenuated PASMCs proliferation and enhanced the expression of contractile genes in vascular smooth muscle cells (VSMCs), while overexpression of miR-9 in normoxia promoted a proliferative phenotype in PASMCs. The primary transcripts of miR-9-1 and miR-9-3, but not miR-9-2, increased dramatically after hypoxia, whereas silencing of the hypoxia-associated transcription factor HIF-1α following hypoxia exposure abolished the enhancement of both primary transcripts in PASMCs. Using in silico analysis, we found three putative HIF-1α binding motifs on miR-9-1 and one motif on miR-9-3 located within the 5-kb region upstream of the transcriptional start sites. Chromatin immunoprecipitation assay revealed that hypoxia enhanced the direct interaction between HIF-1α and the regulatory elements of miR-9-1 and miR-9-3. Reporter assays showed that the regulatory regions of miR-9-1 and miR-9-3 behaved as enhancers in a HIF-1α-dependent manner during hypoxia. Taken together, our data uncover a regulatory mechanism involving HIF-1α-mediated up-regulation of miR-9, which plays a role in the hypoxia-induced phenotypic switch of PASMCs.

Differential regulation of GLUT1 and GLUT8 expression by hypoxia in mammary epithelial cells.

Glucose is a major substrate for milk synthesis and is taken up from the blood by mammary epithelial cells (MECs) through facilitative glucose transporters (GLUTs). The expression levels of GLUT1 and GLUT8 are upregulated dramatically in the mammary gland from late pregnancy through early lactation stages. This study aimed to test the hypothesis that this increase in GLUT1 and GLUT8 expression involves hypoxia signaling through hypoxia inducible factor-1α (HIF-1α) in MECs. Mouse mammary glands showed significantly more hypoxia in midpregnancy through early lactation stages compared with in the virgin stage, as stained by the hypoxia marker pimonidazole HCl. Treatment with hypoxia (2% O2) significantly stimulated glucose uptake and GLUT1 mRNA and protein expression, but decreased GLUT8 mRNA expression in bovine MECs. In MECs, hypoxia also increased the levels of HIF-1α protein in the nuclei, and siRNA against HIF-1α completely abolished the hypoxia-induced upregulation of GLUT1, while having no effect on GLUT8 expression. A 5'-RCGTG-3' core HIF-1α binding sequence was identified 3.7 kb upstream of the bovine GLUT1 gene, and HIF-1α binding to this site was increased during hypoxia. In conclusion, the mammary glands in pregnant and lactating animals are hypoxic, and MECs respond to this hypoxia by increasing GLUT1 expression and glucose uptake through a HIF-1α-dependent mechanism. GLUT8 expression, however, is negatively regulated by hypoxia through a HIF-1α-independent pathway. The regulation of glucose transporters through hypoxia-mediated gene transcription in the mammary gland may provide an important physiological mechanism for MECs to meet the metabolic demands of mammary development and lactation.

HIF-1α Inhibition Reverses Multidrug Resistance in Colon Cancer Cells via Downregulation of MDR1/P-Glycoprotein

BackgroundMultidrug resistance (MDR) is one of the major reasons chemotherapy-based treatments fail. Hypoxia is generally associated with tumor chemoresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) remains unclear. This study aims to explore the molecular mechanisms of reversing colon cancer MDR by focusing on the target gene HIF-1α.MethodsA chemotherapeutic sensitivity assay was used to observe the efficiency of MDR reversal in LoVo multicellular spheroids (MCS). The apoptotic level induced by different drugs was examined by flow cytometry (FCM). Binding of HIF-1α to the MDR1 gene promoter was evaluated by Chromatin immunoprecipitation (ChIP). The relationship between HIF-1α/P-gp expression and sensitivity to chemotherapy was analyzed.ResultsThe sensitivity of LoVo MCS to all four chemotherapy drugs was decreased to varying degrees under hypoxic conditions. After silencing the HIF-1α gene, the sensitivities of LoVo MCS to all four chemotherapy drugs were restored. The apoptotic levels that all the drugs induced were all decreased to various extents in the hypoxic group. After silencing HIF-1α, the apoptosis level induced by all four chemotherapy drugs increased. The expression of HIF-1α and P-gp was significantly enhanced in LoVo MCS after treatment with hypoxia. Inhibiting HIF-1α significantly decreased the expression of MDR1/P-gp mRNA or protein in both the LoVo monolayers and LoVo MCS. The ChIP assay showed that HIF-1α was bound to the MDR1 gene promoter. Advanced colon carcinoma patients with expression of both HIF-1α and P-gp were more resistant to chemotherapy than that with non expression.ConclusionsHIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-gp. The expression of HIF-1α and MDR1/P-gp can be used as a predictive marker for chemotherapy resistance in colon cancer.

PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.

Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy.

Hidden secret in hepatitis B viral X protein mutation and hypoxia-inducible factor-1α in hepatocarcinoma cancer.

Hepatitis B type virus (HBV) is an old hepato oncogenic and hepatitis agent. Hepatitis B viral X protein (HBx)-induced malignant transformation requires the excess amounts of ATP level, inducing the extremely oxygen-deprived condition in the cancer tissues and vessels. To adapt, cells go to shift the hypoxic responsive state by altered hypoxia-responsive molecules such as HIF-1. In addition, tumors avoid or suppress immune recognition in the energy-deprived condition. The hypoxia-inducible factor-1α (HIF-1α) regulates MAP1, histone deacetylase and MAPK pathway. In the hypoxia, the HIF-1α interacts with HIF-1β, allowing DNA binding at the hypoxia response elements (HREs), while HBx binds with the nHLH/PAS domain of HIF-1α, preventing pVHL and HIF-1α binding capacity and degradation of HIF-1α protein. Recent work of Liu et al. [2013] demonstrated that HBx in hepatocellular carcinoma (HCC) tissues contained mutations, affecting the HBx transactivation capacity and C-terminal HBx mutation. In the HCC tissues, the HBx C-terminal mutation and HIF-1α expression were related and the different C-terminal mutations of HBx exhibit the different functionality of HIF-1α. The C-terminal region of amino acids 119-140 was important for the stability and transactivation, and the point mutations K130M/V131I enhance the functionality of HIF-1α, while C-terminal truncation diminish the HIF-1α function.

HIF‐1α Expression as a Protective Strategy of HepG2 Cells Against Fatty Acid‐Induced Toxicity

Free fatty acid-induced lipotoxicity via increased endoplasmic reticulum (ER) stress and hepatocyte apoptosis is a key pathological mechanism of non-alcoholic steatohepatitis. A role of hypoxia-inducible factor 1α (HIF-1α) in this process has been suggested, but direct evidence is lacking. Here, we used HepG2 cells as a model to study whether HIF-1α can reduce palmitic acid-induced lipotoxicity and ER stress. In HepG2 cells treated with 500 µM palmitic acid, HIF-1α expression increased transiently, the decline was associated with increased cleaved caspase-3 expression. Overexpression and knockdown of HIF-1α decreased and exacerbated, respectively, palmitic acid-induced lipoapoptosis. The overexpression also blunted upregulation of the ER stress markers, C/EBP homologous protein (CHOP) and chaperone immunoglobulin heavy chain binding protein (Bip), while the knockdown increased the level of CHOP. In line with this, CHOP promoter activity decreased following HIF-1α binding to the CHOP promoter hypoxia response element. These results indicate that hepatocyte lipotoxicity is associated with decreased HIF-1α expression. It also suggests that upregulation of HIF-1α can be a possible strategy to reduce lipotoxicity in non-alcoholic fatty liver disease.

Inhibition of KAP1 Enhances Hypoxia-Induced Kaposi's Sarcoma-Associated Herpesvirus Reactivation through RBP-Jκ

Hypoxia-inducible factor 1α (HIF-1α) has been frequently implicated in many cancers as well as viral pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. It can stabilize HIF-1α during latent infection and undergoes lytic replication in response to hypoxic stress. However, the mechanism by which KSHV controls its latent and lytic life cycle through the deregulation of HIF-1α is not fully understood. Our previous studies showed that the hypoxia-sensitive chromatin remodeler KAP1 was targeted by the KSHV-encoded latency-associated nuclear antigen (LANA) to repress expression of the major lytic replication and transcriptional activator (RTA). Here we further report that an RNA interference-based knockdown of KAP1 in KSHV-infected primary effusion lymphoma (PEL) cells disrupted viral episome stability and abrogated sub-G1/G1 arrest of the cell cycle while increasing the efficiency of KSHV lytic reactivation by hypoxia or using the chemical 12-O-tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate (NaB). Moreover, KSHV genome-wide screening revealed that four hypoxia-responsive clusters have a high concurrence of both RBP-Jκ and HIF-1α binding sites (RBS+HRE) within the same gene promoter and are tightly associated with KAP1. Inhibition of KAP1 greatly enhanced the association of RBP-Jκ with the HIF-1α complex for driving RTA expression not only in normoxia but also in hypoxia. These results suggest that both KAP1 and the concurrence of RBS+HRE within the RTA promoter are essential for KSHV latency and hypoxia-induced lytic reactivation. Kaposi's sarcoma-associated herpesvirus (KSHV), a DNA tumor virus, is an etiological agent linked to several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). HIF-1α, a key hypoxia-inducible factor, is frequently elevated in KSHV latently infected tumor cells and contributes to KSHV lytic replication in hypoxia. The molecular mechanisms of how KSHV controls the latent and lytic life cycle through deregulating HIF-1α remain unclear. In this study, we found that inhibition of hypoxia-sensitive chromatin remodeler KAP1 in KSHV-infected PEL cells leads to a loss of viral genome and increases its sensitivity to hypoxic stress, leading to KSHV lytic reactivation. Importantly, we also found that four hypoxia-responsive clusters within the KSHV genome contain a high concurrence of RBP-Jκ (a key cellular regulator involved in Notch signaling) and HIF-1α binding sites. These sites are also tightly associated with KAP1. This discovery implies that KAP1, RBP-Jκ, and HIF-1α play an essential role in KSHV pathogenesis through subtle cross talk which is dependent on the oxygen levels in the infected cells.

Hypoxia inducible factor-1α directly regulates nuclear clusterin transcription by interacting with hypoxia response elements in the clusterin promoter.

Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions −806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-1α (HIF-1α) bound directly to these sites and activated transcription. Exposure to the hypoxiamimetic compound CoCl2, incubation under 1% O2 conditions, or overexpression of HIF-1α enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with CoCl2 and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-1α to HRE sites and is epigenetically controlled by methylation of its promoter region.

CD147 Promotes Melanoma Progression Through Hypoxia-Induced MMP2 Activation

Hypoxia enhances MMP2 expression and the invasion and metastatic potential of melanoma cells. CD147 has been shown to induce MMP2 in multiple cancers. To investigate the role of CD147 in hypoxiainduced MMP2 activation, we performed immunohistochemistry (IHC) staining in 206 normal and melanoma tissue samples, and analyzed the correlation between HIF1α and CD147. ChIP (chromosome Immunoprecipitation) in melanoma cell lines supports that HIF1α directly binds to CD147 promoter. Moreover, we made a series of deletion mutants of CD147 promoter, and identified a conserved HIF1α binding site. Point mutation in this site significantly decreased CD147 response to hypoxia. Importantly, knocking down CD147 attenuates MMP2 response to hypoxia in melanoma cell lines. MMP2 could not be efficiently activated by hypoxia in CD147 depletion cells. ELISA data showed that MMP2 secretion was reduced in CD147 depletion cells than control under hypoxia condition. To verify the data from cell culture model, we performed in vivo mouse xenograft experiment. IHC staining showed reduced MMP2 level in CD147 depleted xenografts compared to the control group, with the HIF1α level being comparable. Our study demonstrates a novel pathway mediated by CD147 to promote the MMP2 activation induced by hypoxia, and helps to understand the interplay between hypoxia and melanoma progression.

Key role of endothelial importin-α in VEGF expression and gastric angiogenesis: novel insight into aging gastropathy

Recent in vivo studies demonstrated that aging gastric mucosa has impaired angiogenesis and reduced expression of vascular endothelial growth factor (VEGF). Angiogenesis is triggered by hypoxia and VEGF gene activation, and the latter requires transport of transcription factor(s) into endothelial cell nuclei. We focused on gastric mucosal endothelial cells (GMEC), which are key targets and effectors of gastric angiogenesis, and determined whether and to what extent importin-α, a nuclear transport protein, regulates VEGF gene activation and gastric angiogenesis and the possible role of importin-α in aging gastropathy. GMEC were isolated from rats 3 and 24 mo of age, young (YGEC) and aging (AGEC), respectively. We examined in these cells 1) in vitro angiogenesis, 2) expression of VEGF and importin-α, 3) nuclear transport of hypoxia-inducible factor (HIF)-1α by importin-α, 4) binding of HIF-1α to the VEGF gene promoter, and 5) effects of importin-α silencing in YGEC and its upregulation in AGEC on angiogenesis and VEGF expression. AGEC exhibited significantly impaired in vitro angiogenesis by fourfold and decreased expression of VEGF, importin-α, and nuclear HIF-1α by 1.4-fold, 1.6-fold, and 2.9-fold, respectively, vs. YGEC. Upregulation of importin-α in AGEC significantly reversed all these abnormalities. In YGEC, knockdown of importins-α1 and -α3 significantly reduced in vitro angiogenesis by 93% and 73% and VEGF expression by 48% and 52%, respectively. The above findings demonstrate that importin-α is a novel and critical regulator of gastric angiogenesis. Its reduced expression in AGEC is the key mechanism for impaired angiogenesis and reduced VEGF.

Hypoxia Regulates the Expression of the Neuromedin B Receptor through a Mechanism Dependent on Hypoxia-Inducible Factor-1α

The neuromedin B receptor (NMB-R), a member of the mammalian bombesin receptor family, is frequently overexpressed in various tumors. In the present study, we found that exposure to hypoxic conditions increases the levels of NMBR mRNA and protein in breast cancer cells, which are tightly regulated by hypoxia-inducible factor-1α (HIF-1α). We confirmed the effect of HIF-1α on NMBR transcription by performing an NMBR promoter-driven reporter assay and then identified a functional hypoxia-responsive element (HRE) in the human NMBR promoter region. Further, the binding of HIF-1α to the NMBR promoter was corroborated by electrophoretic mobility shift and chromatin immunoprecipitation assays, which showed that HIF-1α specifically and directly bound to the NMBR promoter in response to hypoxia. Immunohistochemical analysis of a xenograft and a human breast cancer tissue array revealed a significant correlation between NMB-R and HIF-1α expression. Taken together, our findings indicate that hypoxia induces NMB-R expression through a novel mechanism to regulate HIF-1α expression in breast cancer cells.

A perylene derivative regulates HIF-1α and Stat3 signaling pathways

It is becoming increasingly evident that improving the cure rate of many cancers will require treatment regimens hit more than one validated tumor targets. Developing an anti-cancer agent that targets two oncoproteins simultaneously is a promising strategy for accomplishing this goal. It would be expected to promote drug efficacy, reduce therapy-resistant without introducing additional toxic side effects. HIF-1α is a key regulator of the cellular response to hypoxia and is involved in tumor angiogenesis and cancer cell survival, glucose metabolism, and invasion. Stat3 has several oncogenic functions, including suppression of anti-tumor immune responses and promotion of inflammation. Recently, we have identified the perylene derivative, TEL03, as a dual inhibitor that targets both HIF-1α and Stat3. TEL03 blocks the expression of both HIF-1α and Stat3, regulated oncogenes (e.g., Bcl-2, VEGF, Glut1, and others) in cancer cells, and induces cancer cell apoptosis. The results demonstrated that: (i) TEL03 blocks Stat3 phosphorylation, and inhibits Stat3 transcriptional activity; and (ii) interferes the binding of HIF-1α to p300/CBP inducing its degradation by proteasomes under hypoxic conditions. Our in vivo tests showed that as a dual inhibitor, TEL03 dramatically inhibited tumor growth, and provided the evidence that targeting both HIF-1α and Stat3 simultaneously could be a promising strategy for breast and pancreatic cancer therapies.

Role of hypoxia inducible factor-1α in the regulation of the cancer-specific variant of organic anion transporting polypeptide 1B3 (OATP1B3), in colon and pancreatic cancer

Organic anion transporting polypeptide 1B3 (OATP1B3) was initially considered to be a liver-specific transporter, mediating the uptake of a variety of endogenous and xenobiotic substances. Over the past decade, several investigations reported that OATP1B3 is also expressed across multiple types of cancers. Only recently, our laboratory and others demonstrated the identity of cancer-specific OATP1B3 variants (csOATP1B3) arising from the use of an alternative transcription initiation site, different from the wildtype (WT) OATP1B3 expressed in the normal liver. However, the mechanisms regulating the expression of csOATP1B3 remained unknown. In our current study, we investigated the role of hypoxia and the involvement of hypoxia inducible factor-1α (HIF-1α) in regulating the transcription of csOATP1B3. Our RT-PCR and immunoblotting results indicated that csOATP1B3, but not WT OATP1B3, can be induced in response to ambient or chemical hypoxia (upon exposure to 1% O2 or cobalt chloride). Reporter assays with deletion and mutated constructs of the csOATP1B3 promoter revealed a functional hypoxia response element (HRE) located in the proximal upstream region. Constructs harboring the HRE displayed the upregulated reporter gene expression in response to hypoxia, but not when mutated. Electrophoretic mobility shift assays using a biotin-labeled csOATP1B3 promoter HRE probe indicated the binding of HIF-1α, which was blocked by an excess of unlabeled csOATP1B3 probe. Furthermore, siRNA-based knockdown of HIF-1α caused a substantial decrease in the expression level of csOATP1B3. Taken together, these findings demonstrate that the transcription of csOATP1B3 is actively engaged during hypoxia, through a commonly utilized pathway involving HIF-1α.

Melittin Suppresses HIF-1α/VEGF Expression through Inhibition of ERK and mTOR/p70S6K Pathway in Human Cervical Carcinoma Cells

ObjectiveMelittin (MEL), a major component of bee venom, has been associated with various diseases including arthritis, rheumatism and various cancers. In this study, the anti-angiogenic effects of MEL in CaSki cells that were responsive to the epidermal growth factor (EGF) were examined.Methodology/Principal FindingsMEL decreased the EGF-induced hypoxia-inducible factor-1α (HIF-1α) protein and significantly regulated angiogenesis and tumor progression. We found that inhibition of the HIF-1α protein level is due to the shortened half-life by MEL. Mechanistically, MEL specifically inhibited the EGF-induced HIF-1α expression by suppressing the phosphorylation of ERK, mTOR and p70S6K. It also blocked the EGF-induced DNA binding activity of HIF-1α and the secretion of the vascular endothelial growth factor (VEGF). Furthermore, the chromatin immunoprecipitation (ChIP) assay revealed that MEL reduced the binding of HIF-1α to the VEGF promoter HRE region. The anti-angiogenesis effects of MEL were confirmed through a matrigel plus assay.ConclusionsMEL specifically suppressed EGF-induced VEGF secretion and new blood vessel formation by inhibiting HIF-1α. These results suggest that MEL may inhibit human cervical cancer progression and angiogenesis by inhibiting HIF-1α and VEGF expression.

In silico investigation of PHD-3 specific HIF1-α proline 567 hydroxylation: A new player in the VHL/HIF-1α interaction pathway?

Hypoxia inducible factor 1α (HIF-1α) regulates oxygen homeostasis in the cell through a sensing mechanism involving its hydroxylation and binding to the von Hippel–Lindau (VHL) tumor suppressor. This mechanism is mediated through hydroxylation of HIF-1α proline 564, although in vitro tests have previously shown an alternative hydroxylation at proline 567 by PHD-3. Here, molecular dynamics simulations were used to investigate the structural effect of this alternative hydroxylation. A specific hydrogen bond network rearrangement and improved electrostatic energy for hydroxylated P567 are compatible with an increase in HIF-1α binding affinity. Sequence analysis also confirms P567 to be vastly conserved during evolution, indicating a possible role for this alternative, PHD-3 driven, post translational modification in pVHL–HIF-1α complex formation.

An Undesired Effect of Chemotherapy: GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT, NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

Hypoxia-induced regulation of the very low density lipoprotein receptor

The very low density lipoprotein receptor (VLDLr) is highly upregulated during hypoxia in mouse cardiomyocytes and in human and mouse ischemic hearts causing a detrimental lipid accumulation. To know how the gene is regulated is important for future studies. In this study, we have thoroughly mapped the 5′-flanking region of the mouse VLDLr promoter and show that the hypoxia-mediated increase in VLDLr expression is dependent on Hif-1α binding to a hypoxia responsive element (HRE) located at −162 to −158bp 5′of translation start. We show that classical HRE sites and the previously described PPARγ and Sp1 binding are not involved in the hypoxia-induced regulation of the VLDLr promoter. Using a chromatin immunoprecipitation (ChIP) assay, we show that Hif-1α specifically binds and activates the mouse VLDLr promoter at the previously described non-classical HRE in HL-1 cells. We also show that the same HRE is present and active in response to hypoxia in human cardiomyocytes, however at a different location (−812bp from translation start). These results conclude that in the hypoxic hearts of mice and men, the VLDLr gene is regulated by a direct binding of Hif-1α to the VLDLr promoter.

PWE-159 Hif-1Alpha-Dependent Gastrin Gene Expression Mediates Resistance to Hypoxia-Inducible Apoptosis in a Human Colorectal Cancer Cell Line

IntroductionUnderstanding the molecular processes mediating colorectal cancer (CRC) tumorigenesis will enable the development of targeted therapies that selectively disrupt the pathways responsible for tumour growth. The gastrin family of growth...