Normal Oxygen Conditions Research Articles

Role of HLA-G1 in trophoblast cell proliferation, adhesion and invasion

Trophoblast cells are important in embryo implantation and fetomaternal tolerance. HLA-G is specifically expressed at the maternal–fetal interface and is a regulator in pregnancy. The aim of the present study was to detect the effect of HLA-G1 on trophoblast cell proliferation, adhesion, and invasion. Human trophoblast cell lines (JAR and HTR-8/SVneo cells) were infected with HLA-G1-expressing lentivirus. After infection, HLA-G1 expression of the cells was detected by western blotting. Cell proliferation was detected by the BrdU assay. The cell cycle and apoptosis of JAR and HTR-8/SVneo cells was measured by flow cytometry (FCM). The invasion of the cells under different conditions was detected by the transwell invasion chamber assay. HLA-G1 didn't show any significant influence on the proliferation, apoptosis, adhesion, and invasion of trophocytes in normal culture conditions. However, HLA-G1 inhibited JAR and HTR-8/SVneo cells invasion induced by hepatocyte growth factor (HGF) under normal oxygen conditions. In conditions of hypoxia, HLA-G1 couldn't inhibit the induction of cell invasion by HGF. HLA-G1 is not an independent factor for regulating the trophocytes. It may play an indirect role in embryo implantation and formation of the placenta.

An initial study of the influences of oxygen conditions on wild-type magnetotactic bacteria in sediment

Magnetotactic bacteria (MTB) orient and migrate along geomagnetic field lines in a process known as magnetotaxis, while their swimming direction is determined by flagella rotation (clockwise or counterclockwise) in response to surrounding chemical conditions, i.e., chemotaxis. The combination of magnetotaxis and chemotaxis (magneto-chemotaxis) is expected to govern MTB behaviors and vertical distribution in chemically stratified sediment. Magneto-aerotaxis is a process in which oxygen concentration determines MTB swimming direction. However, direct validation of magneto-aerotaxis in sediment, where MTB primarily occur, has been limited to date. Such validation is crucial because low alignment degree and complex chemical conditions in sediment are not comparable to those in the aqueous environment. Herein, specifically designed experiments were conducted to investigate the effects of oxygen conditions on two wild-type MTB in sediment, rod-shaped Candidatus Magnetobacterium bavaricum ( M. bavaricum ) and unspecified cocci collected from Chiemsee, a freshwater lake in southern Germany. In the first experiment, oxygen gradient development and MTB vertical distribution were closely monitored during reformation of new sediment stratification. The MTB population peaked immediately below the sediment-water interface, which was likely because of magneto-aerotaxis. However, there were no observable changes in oxic-anoxic interface (OAI), and MTB distribution later extended to greater depth, indicating other driving mechanisms. A second experiment was conducted to determine if north-seeking (NS) MTB become south-seeking (SS) when moved into anoxic conditions; however, this conversion was not detected during oxygen concentration reduction. The magneto-aerotaxis of the two examined MTB were not directly validated. Long-term stable anoxic conditions led to MTB distribution downward, which is consistent with the distribution under normal oxygen conditions, indicating MTB adaptation to the environment. These findings indicate that oxygen conditions likely play a role in MTB behaviors and vertical distribution in sediment, but that they do not act as directly influencing or critical factors.

Low glucose microenvironment of normal kidney cells stabilizes a subset of messengers involved in angiogenesis.

As glucose is a mandatory nutrient for cell proliferation and renewal, it is suspected that glucose microenvironment is sensed by all cell types to regulate angiogenesis. Several glucose‐sensing components have been partially described to respond to high glucose levels. However, little is known about the response to low glucose. Here, we used well‐differentiated isolated normal rat renal tubules under normal oxygenation conditions to assess the angiogenic response to low glucose. In apparent paradox, but confirming observations made separately in other models, high glucose but also low glucose increased mRNA level of vascular endothelial growth factor A (VEGFA). A subset of mRNAs including hypoxia‐inducible factor 1A (HIF1A), angiopoietin receptor (TIE‐2), and VEGF receptor 2 (FLK1) were similarly glucose‐sensitive and responded to low glucose by increased stability independently of HIF1A and HIF2A proteins. These results contribute to gain some insights as to how normal cells response to low glucose may play a role in the tumor microenvironment.

Abstract 977: Characterizing the regulation and function of miR-155 in hypoxia biology

Abstract Aberrant expression of microRNAs has been correlated with the initiation and progression of many diseases. One such microRNA, miR-155, is an oncogenic microRNA that has been associated with several different types of cancer, including lung, breast, and lymphoma. We are studying the role of miR-155 in promoting hypoxia-driven tumor phenotypes such as therapy resistance and genomic instability. Previously, we have shown that miR-155 expression is induced under hypoxic conditions and that over-expression of miR-155 under normal oxygen conditions confers resistance to ionizing radiation therapy. Additionally, we have shown that by knocking down expression of miR-155 under hypoxic conditions, we were able to sensitize these cells to ionizing radiation. Here, we demonstrate that miR-155 expression is tightly linked to genomic instability, another characteristic of hypoxia. We have found that over-expression of miR-155 drives increased mutation frequency as measured by a mutation reporter assay screen both in vitro and in vivo. These results are consistent with a recent finding that miR-155 over-expression targets mismatch repair proteins, MLH1, MSH2, and MSH6. An increase in mutation frequency could be due to a decrease in mismatch repair function. Surprisingly, we have also found that loss of miR-155 increases the baseline level of double strand breaks observed in a fibroblast cell line. Taken together, this data suggests that a finite regulation of miR-155 is necessary to maintain cellular homeostasis. Our goal is to gain a better understanding of the role of miR-155 in promoting hypoxic cancer cell survival. Citation Format: Jennifer Czochor, Peter M. Glazer. Characterizing the regulation and function of miR-155 in hypoxia biology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 977. doi:10.1158/1538-7445.AM2014-977

Ghrelin increases lymphocytes in chronic normobaric hypoxia.

Hypoxia is a condition of decreased availability of oxygen. To adapt hypoxia, some changes in blood cells occur in the body. The aim of this study was to evaluate the effect of ghrelin on different types of blood cell in normobaric hypoxia situation. Thirty-two animals were divided in 4 groups (n=8): control (C), ghrelin (G), hypoxia (H), and hypoxic animals that received ghrelin (H+G). Hypoxia (11%) was induced by an Environmental Chamber System GO2 Altitude. Animals in ghrelin groups received a subcutaneous injection of ghrelin (150 &mu;g/kg/day) for 14 days. Our results show that ghrelin significantly (p<0.05) increased RBC and Hct levels, whereas it significantly (p<0.05) decreased lymphocytes in the blood. RBC, Hct, Hb concentration, platelet and MCV increased significantly (p<0.05) in hypoxic conditions but lymphocytes, monocytes and Polymorphonuclears did not show any significant changes. Platelets had a significant (p<0.05) decrease in hypoxic conditions and ghrelin administration in hypoxic conditions could increase lymphocyte levels significantly (p<0.05). Effect of ghrelin on blood cells could be related to blood oxygen level. Ghrelin in normal oxygen conditions increases RBC and Hct levels but decreases lymphocytes, whereas in hypoxic conditions, ghrelin increases blood lymphocytes.

Methylprednisolone inhibits the proliferation and affects the differentiation of rat spinal cord-derived neural progenitor cells cultured in low oxygen conditions by inhibiting HIF-1α and Hes1 in vitro.

Although there is much controversy over the use of methylprednisolone (MP), it is one of the main drugs used in the treatment of acute spinal cord injury (SCI). The induction of the proliferation and differentiation of endogenous neural progenitor cells (NPCs) is considered a promising mode of treatment for SCI. However, the effects of MP on spinal cord-derived endogenous NPCs in a low oxygen enviroment remain to be delineated. Thus, the aim of this study was to investigate the potential effects of MP on NPCs cultured under low oxygen conditions in vitro and to elucidate the molecular mechanisms involved. Fetal rat spinal cord-derived NPCs were harvested and divided into 4 groups: 2 groups of cells cultured under normal oxygen conditions and treated with or without MP, and 2 groups incubated in 3% O2 (low oxygen) treated in a similar manner. We found that MP induced suppressive effects on NPC proliferation even under low oxygen conditions (3% O2). The proportion of nestin-positive NPCs decreased from 51.8±2.46% to 36.17±3.55% following the addition of MP and decreased more significantly to 27.20±2.68% in the cells cultured in 3% O2. In addition, a smaller number of glial fibrillary acidic protein (GFAP)-positive cells and a greater number of microtubule-associated protein 2 (MAP2)-positive cells was observed following the addition of MP under both normal (normoxic) and low oxygen (hypoxic) conditions. In response to MP treatment, hypoxia-inducible factor-1α (HIF-1α) and the Notch signaling pathway downstream protein, Hes1, but not the upstream Notch-1 intracelluar domain (NICD), were inhibited. After blocking NICD with a γ-secretase inhibitor (DAPT) MP still inhibited the expression of Hes1. Our results provide insight into the molecular mechanisms responsible for the MP-induced inhibition of proliferation and its effects on differentiation and suggest that HIF-1α and Hes1 play an important role in this effect.

Direct phosphorylation events involved in HIF-&amp;alpha; regulation: the role of GSK-3&amp;beta;

Hypoxia-inducible factors (HIFs), consisting of α- and β-subunits, are critical regulators of the transcriptional response to hypoxia under both physiological and pathological conditions. To a large extent, the protein stability and the recruitment of coactivators to the C-terminal transactivation domain of the HIF α-subunits determine overall HIF activity. The regulation of HIF α-subunit protein stability and coactivator recruitment is mainly achieved by oxygen-dependent posttranslational hydroxylation of conserved proline and asparagine residues, respectively. Under hypoxia, the hydroxylation events are inhibited and HIF α-subunits stabilize, translocate to the nucleus, dimerize with the β-subunits, and trigger a transcriptional response. However, under normal oxygen conditions, HIF α-subunits can be activated by various growth and coagulation factors, hormones, cytokines, or stress factors implicating the involvement of different kinase pathways in their regulation, thereby making HIF-α-regulating kinases attractive therapeutic targets. From the kinases known to regulate HIF α-subunits, only a few phosphorylate HIF-α directly. Here, we review the direct phosphorylation of HIF-α with an emphasis on the role of glycogen synthase kinase-3β and the consequences for HIF-1α function.

Emerging evidence of the physiological role of hypoxia in mammary development and lactation

Hypoxia is a physiological or pathological condition of a deficiency of oxygen supply in the body as a whole or within a tissue. During hypoxia, tissues undergo a series of physiological responses to defend themselves against a low oxygen supply, including increased angiogenesis, erythropoiesis, and glucose uptake. The effects of hypoxia are mainly mediated by hypoxia-inducible factor 1 (HIF-1), which is a heterodimeric transcription factor consisting of α and β subunits. HIF-1β is constantly expressed, whereas HIF-1α is degraded under normal oxygen conditions. Hypoxia stabilizes HIF-1α and the HIF complex, and HIF then translocates into the nucleus to initiate the expression of target genes. Hypoxia has been extensively studied for its role in promoting tumor progression, and emerging evidence also indicates that hypoxia may play important roles in physiological processes, including mammary development and lactation. The mammary gland exhibits an increasing metabolic rate from pregnancy to lactation to support mammary growth, lactogenesis, and lactation. This process requires increasing amounts of oxygen consumption and results in localized chronic hypoxia as confirmed by the binding of the hypoxia marker pimonidazole HCl in mouse mammary gland. We hypothesized that this hypoxic condition promotes mammary development and lactation, a hypothesis that is supported by the following several lines of evidence: i) Mice with an HIF-1α deletion selective for the mammary gland have impaired mammary differentiation and lipid secretion, resulting in lactation failure and striking changes in milk compositions; ii) We recently observed that hypoxia significantly induces HIF-1α-dependent glucose uptake and GLUT1 expression in mammary epithelial cells, which may be responsible for the dramatic increases in glucose uptake and GLUT1 expression in the mammary gland during the transition period from late pregnancy to early lactation; and iii) Hypoxia and HIF-1α increase the phosphorylation of signal transducers and activators of transcription 5a (STAT5a) in mammary epithelial cells, whereas STAT5 phosphorylation plays important roles in the regulation of milk protein gene expression and mammary development. Based on these observations, hypoxia effects emerge as a new frontier for studying the regulation of mammary development and lactation.

Changes in pulmonary gas exchange during resistive loading that do not cause a sense of shortness of breath

It is known that hypoxic hypometabolism occurs as a result of reduced delivery of oxygen to tissues. We have shown earlier that this response may be observed during latent hypoxia, when blood oxygen saturation (SpO2) is reduced to 95%. There is a hypothesis that the body can prevent a disturbance of the balance between oxygen delivery and consumption by developing a protective response to reduce oxygen demand. To test the hypothesis, a study was carried out under normal oxygenation conditions, in which a weak additional resistance of 0.4 cm H2O L−1 s was used as a trigger. The study sample comprised 14 healthy subjects of both sexes. Gas exchange parameters under normal and combined resistive loading conditions were studied. It was found that the respiratory rate was reduced by 8%, and the tidal volume was increased by 12%. At the same time, oxygen consumption and carbon dioxide production were significantly decreased by 9 and 10%, respectively. These findings suggest that a decrease in the rates of oxygen consumption and carbon dioxide production is a nonspecific protective response to prevent the disturbance of the balance between oxygen delivery and consumption.

Effects of hypoxia on proliferation and osteogenic differentiation of periodontal ligament stem cells: an in vitro and in vivo study.

Changes in oxygen concentration may influence various innate characteristics of stem cells. The effects of varying oxygen concentration on human periodontal ligament stem cells (HPDLSCs) has not been explored, particularly under hypoxia-related conditions. First, HPDLSCs were cultured from the periodontium of human teeth using the outgrowth method. STRO-1 and CD146 expression of HPDLSCs was investigated by flow cytometry. To detect the multilineage differentiation capacities of HPDLSCs, osteogenic-like and adipogenic-like states were induced in cells. Next, HPDLSCs (passage 3) were exposed to normal oxygen (21% O2) or hypoxia (2% O2) conditions for 7 days and cell proliferation was evaluated. After culture in osteogenic medium for 7 days, osteoblastic differentiation was evaluated by semi-quantitative reverse transcription-polymerase chain reaction analysis to detect 3 osteoblastic markers: core-binding factor a 1/runt-related transcription factor 2, osteocalcin, and osteopontin. In addition, each cell group was incubated with a hydroxyapatite/tricalcium phosphate carrier and transplanted subcutaneously into the back of immunocompromised mice to investigate transplantation differences in vivo. HPDLSCs were isolated, cultured, and successfully identified. After exposure of HPDLSCs to hypoxia for 7 days, the proliferation rate was increased and showed higher osteogenic differentiation potential compared to control cells. After 12 weeks of transplantation, hypoxia-treated HPDLSCs differentiated into osteoblast-like cells that formed bone-like structures. These results suggest that oxygen concentrations affect various aspects of HPDLSC physiology and that hypoxia enhances osteogenic differentiation both in vivo and in vitro. Oxygen concentration may be a critical parameter for HPDLSCs during expansion and differentiation.

Targeting cisplatin-resistant human tumor cells with metabolic inhibitors

Although cisplatin is the drug of choice in treating lung cancer patients, relapse and resistance is a common drawback to its clinical effectiveness. Based on cisplatin's reported ability to interfere with numerous cellular components, including mitochondria, we probed alterations in metabolism in cisplatin-resistant tumor cell lines to reveal targets for overcoming this important form of resistance. Cisplatin-resistant lung and ovarian cancer cell lines were used to evaluate the efficacy of metabolic inhibitors for selectively targeting cisplatin-resistant cells under varying oxygen conditions. Three cisplatin-resistant cancer cell lines expressed lower HKII protein when compared to the respective cisplatin-sensitive cancer cell lines from which they were derived. Under anaerobic and hypoxic conditions, treatment with the glycolytic inhibitors 2-deoxyglucose (2-DG) and 2-fluorodeoxyglucose (2-FDG) correlated with increased cytotoxicity and more pronounced decreases in lactate production in cisplatin-resistant cells, indicating a greater blockage of glycolysis. Knockdown of HKI or HKII with siRNA in the parental lung cancer cell lines led to increased 2-FDG-induced cell death under anaerobic conditions. Under normal oxygen conditions, blockage of either fatty acid oxidation or deprivation of glutamine resulted in cell death in cisplatin-resistant lung cancer cell lines. Altered hexokinase levels in cisplatin-resistant cancer cell lines leads to increased sensitivity to glycolytic inhibition under anaerobic conditions, whereas under normoxic conditions, blockage of either fatty acid oxidation or deprivation of glutamine leads to cell death. These findings may be clinically applicable when considering cisplatin resistance.

Impaired glucose homeostasis after a transient intermittent hypoxic exposure in neonatal rats

This initial report presents a neonatal rat model with exposure to a transient intermittent hypoxia (IH), which results in a persisting diabetes-like condition in the young rats. Twenty-five male pups were treated at postnatal day 1 with IH exposure by alternating the level of oxygen between 10.3% and 20.8% for 5h. The treated animals were then maintained in normal ambient oxygen condition for 3week and compared to age-matched controls. The IH treated animals exhibited a significantly higher fasting glucose level than the control animals (237.00±19.66mg/dL vs. 167.25±2.95mg/dL; P=0.003); and a significantly lower insulin level than the control (807.0±72.5pg/mL vs. 1839.8±377.6pg/mL; P=0.023). There was no difference in the mass or the number of insulin producing beta cells as well as no indicative of inflammatory changes; however, glucose tolerance tests showed a significantly disturbed glucose homeostasis. In addition, the amount of C-peptide secreted from the islets harvested from the IH animals were decreased significantly (from 914pM in control to 809pM in IH; P=0.0006) as well. These observations demonstrate that the neonatal exposure to the IH regimen initiates the development of deregulation in glucose homeostasis without infiltration of inflammatory cells.

Comparison of Hemoglobins from Various Subjects Living in Hypoxia

The aim of this research was to obtain the different characteristics of haemoglobin molecules in subjects under hypoxic condition, namely eel, catfish, suckermouth fish, green sea turtle using an electrophoresis technique. We used human umbilical cord blood and thalassemia patient blood, as well as a normal adult-human blood as controls. The proteins obtained after electrophoresis process were stained with two different colouring techniques, each based on different principles. Both staining techniques gave practically identical results. Subject that live in hypoxic condition has a different haemoglobin in comparison to the one found in adult human live in normal oxygen condition (normoxia). These hypoxia-adapted or -needed hemoglobin migrate slower than adult human hemoglobin from normoxia. This observation suggests that hemoglobin which is needed to live in hypoxic condition or environment is a different molecule. Whether this hemoglobin from hypoxic condition has a higher affinity to oxygen is not yet known. Further study is needed to clarify this issue.

Abstract 1030: The intriguing interplay between EGFR inhibitors and the hypoxic microenvironment: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines.

Abstract Introduction: Since the epidermal growth factor receptor (EGFR) is overexpressed in more than 90% of all head and neck squamous cell carcinomas (HNSCC) and activation of EGFR initiates important signal transduction pathways in carcinogenesis, it has emerged as a promising therapeutic target. Nevertheless, one main challenge alongside the treatment with EGFR inhibitors remains, being drug resistance. An important potential mechanism involves the hypoxic tumor microenvironment. As our knowledge about the exact impact of tumor hypoxia on the activity of EGFR inhibitors and vice versa remains limited, the present study investigated the cytotoxic effect of the EGFR-targeting monoclonal antibody cetuximab and the EGFR tyrosine kinase inhibitor erlotinib under normoxic versus hypoxic conditions. Materials &amp; Methods: Three cetuximab-sensitive HNSCC cell lines (SC263, LICR-HN2 and LICR-HN5) were treated either with 0 - 10 nM cetuximab for 168 hours or with 0 - 20 μM erlotinib for 72 hours. Cells were incubated under normal or reduced oxygen conditions for 24 or 72 hours immediately after addition of the drug, with hypoxia (&amp;lt;0.1% O2) being achieved in a Bactron IV anaerobic chamber. Cell survival was assessed with the sulforhodamine B assay and IC50 values (representing the drug concentration reducing cell growth to 50%) were calculated. Results: Cetuximab and erlotinib establish a dose-dependent growth inhibition under both normal and reduced oxygen conditions in all three HNSCC cell lines. Considering the effect of cetuximab on cell survival, no significant differences were observed in IC50 values when cells were incubated under normoxia versus hypoxia for 24 or 72 hours (p=0.790, p=0.807 and p=0.288 for SC263, LICR-HN2 and LICR-HN5 respectively). For example, in LICR-HN2 HNSCC cells, cetuximab treatment resulted in IC50 values of 0.14±0.09 nM, 0.19±0.12 nM, and 0.15±0.07 nM for incubation under normoxia versus hypoxia for 24 or 72 hours, respectively. Similarly, the efficacy of erlotinib was not influenced by the oxygen tension in LICR-HN2 and LICR-HN5 cells (p=0.171 and p=0.387, respectively). In SC263 cells, however, a significantly increased sensitivity to erlotinib (p=0.006) was shown when cells were incubated under hypoxic conditions, with IC50 values of 1.53±1.07 μM, 0.81±0.28 μM, and 0.40±0.00 μM for incubation under normoxic conditions versus hypoxic conditions for 24 hours or 72 hours, respectively. Conclusion: Our results suggest that, in the HNSCC cell lines included in our study, both EGFR-inhibitors cetuximab and erlotinib maintain their growth inhibitory effect under hypoxia. As poor oxygenation is a pathophysiological property of the majority of human solid tumors, including HNSCC, further studies are warranted to define the precise mechanistic and therapeutic implications of these observations. Citation Format: Carolien Boeckx, Jolien Van den Bossche, Marc Baay, Vanessa Deschoolmeester, Pol Specenier, Olivier Vanderveken, Danielle Van den Weyngaert, Jan B. Vermorken, Marc Peeters, Filip Lardon, An Wouters. The intriguing interplay between EGFR inhibitors and the hypoxic microenvironment: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1030. doi:10.1158/1538-7445.AM2013-1030

Doublecortin-like kinase 1 exhibits cancer stem cell-like characteristics in a human colon cancer cell line.

Colon cancer stem cells (CSCs) are implicated in colorectal cancer carcinogenesis, metastasis, and therapeutic resistance. The identification of these cells could help to develop novel therapeutic strategies. Doublecortin-like kinase 1 (DCLK1) has been viewed as a marker for gastrointestinal stem cells that fuel the self-renewal process, however others view them as a marker of Tuft cells or as an enteroendocrine subtype. The purpose of this study was to use a colon cancer cell line to identify and characterize the stem-like characteristics of the DCLK1+ cell population. To enrich stem-like cells, HCT116 cells (derived from colon adenocarcinomas) were cultured using serum-free media to form spheres under both normal oxygen and hypoxia condition. DCLK1 transcript expression in the adherent parental cells and spheroids was quantified using quantitative real time reverse transcription- polymerase chain reaction [(q)RT-PCR]. DCLK1 protein expression was determined using flow cytometry. Self-renewal capability from adherent parental cells and spheroids was determined using extreme limiting dilution analysis (ELDA). Under both normal oxygen and hypoxia condition, the adherent parental cells were composed of cells that express low levels of DCLK1. However, spheroids exhibited an increased frequency of cells expressing DCLK1 on both mRNA and protein levels. Cells derived from spheroids also possess stronger self-renewal capability. The higher fraction of DCLK1+ cells exhibited by spheroids and hypoxia reflects the stem-like characteristics of these cells. DCLK1 may represent an ideal marker to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

Effects of suppression hypoxia-inducible factor-1α expression by small interfering RNA on glycolysis inesophageal carcinoma

Objective To investigate the effects of hypoxia-inducible factor （HIF）-1α on glycolysis of human esophageal squamous carcinoma cells and the possible mechanism. Methods TEl3 and Ecal09 cells were cultured under normal oxygen （20%O2 ） and hypoxia （1%O2 ） conditions. The hypoxia was duration 6 hours, 12 hours, 24 hours and 48 hours. HIF-1α gene was stable silented by RNA interference method and TE13/small interfering HIF cells and Ecal09/siHIF cells were obtained. The cell culture condition and time was same as TEl3 and Ecal09 cells. The changes of HIF-1α expression were detected by Western-blot. The changes of lactic acid concentration in cellculture supernatant were determined by Spectrophotometry. The changes of glucose transporter-1 （GLUT-l） and lactic dehydrogenase A （LDHA） expression at mRNA level were examined by real- time polymerase chain reaction. The changes of GLUT-1 and LDHA expression at protein level were tested by Western blot. Using t or t＇ test to analyze the effects of hypoxia duration on HIF-1α expression at protein level. One-way ANOVA was applied for the difference analysis between the groups. Results In TEl3 and Ecal09 cells, the HIF-1α expression significantly increased under hypoxia condition and reached the peak at 12 hour （t=6. 11, 8.31; both P〈0.05）. The lactic acid secretion of TEl3/siHIF cells and Ecal09/siHIF cells was （1.24±0.33） and （1.28±0.37） mmol/L, which significantly decreased when compared with TEl3 and Ecal09 cells [（3.25~ 1.34） and （4.91± 1.69） mmol/L, t=2.53, 3.59, both P〈0. 051. The lactic acid secretion of TEl3 and Ecal09 ceils significantly increased after hypoxia [（6.48±1.73） and （8.02± 1.95） retool/L, t= 2. 715, 2. 050, both P〈0.05]. There was no significant lactic acid secretion in TE13/siHIF cells and Ecal09/siHIF cells after hypoxia （P〉 0.05）. The expressions of GLUT-1 and LDHA at mRNA level were significantly suppressed in TEl3/siHIF cells and Ecal09/siHIF cells （normal oxygen., t= 6.98, 3.92, 7.25, 3.67, all P〈0. 05）. The expression of GLUT-1 at protein level remarkably weaked （normal oxygen., t=4.57,16.56, hypoxia：t=6. 19,6.09,all P〈0.05）, while the expression of LDHA at protein level slightly decreased （P〉0. 05）. Conclusions The level of glycolysis can be lowered by suppression HIF-1α expression in human esophageal squamous carcinoma cells. The pathway may be involved in the suppression of GLUT-1 and LDHA expression. Except for HIF-1α, there may be other regulating factors in LDHA protein expression at same time. Key words: Hypoxia-inducible factor 1, alpha subunit; Glycolysis; RNA, small interfering; Esophageal neoplasms

Evaluation of the effects of nicorandil and its molecular precursor (without radical NO) on proliferation and apoptosis of 786-cell

Nicorandil is a nitric oxide (NO) donor used in the treatment of angina symptoms. It has also been reported to protect cells and affect the proliferation and death of cells in some tissues. The molecules that interfere with these processes can cause dysfunction in healthy tissues but can also assist in the therapy of some disorders. In this study we examined the effect of nicorandil and of the molecular precursor that does not have the NO radical (N-(beta-hydroxyethyl) nicotinamide) on the cell proliferation and death of human renal carcinoma cells (786-O) under normal oxygenation conditions. The molecular precursor was used in order to analyze the effects independents of NO. In the cytotoxicity test, nicorandil was shown to be cytotoxic at very high concentrations and it was more cytotoxic than its precursor (cytotoxic at concentrations of 2,000 and 3,000μg/mL, respectively). We propose that the lower cytotoxicity of the precursor is due to the absence of the NO radical. In this study, the cells exposed to nicorandil showed neither statistically significant changes in cell proliferation nor increases in apoptosis or genotoxicity. The precursor generated similar results to those of nicorandil. We conclude that nicorandil causes no changes in the proliferation or apoptosis of the cell 786-O in normal oxygenation conditions. Moreover, the lack of NO radical in the precursor molecule did not show a different result, except in the cell cytotoxicity.

Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Random integration is a phenomenon in which transfected DNA molecules integrate into (random sites of) the host genome via non-homologous recombination. Although it is assumed that repair of DNA double-strand breaks leads to random integration events, how these endogenous DNA lesions are generated in living cells is poorly understood. In this study, we present evidence that DNA topoisomerase IIa (Top2α) and reactive oxygen species (ROS) are responsible for causing genomic DNA damage that leads to random integration. Specifically, we employed a human pre-B lymphocyte cell line to examine the effects of cellular Top2 expression levels and oxygen concentrations during cell culture. We find that treating cells with Top2α siRNA significantly reduces random integration frequency, while the absence of Top2β had little or no impact. We also show that cells continuously cultured under low (3%) oxygen culture conditions after electroporation display reduced random integration frequency compared to that under normal (21%) oxygen conditions. These findings support the notion that Top2α protein and ROS are endogenous factors that can produce DNA damage leading to random integration of transfected DNA in human cells.

Modulation of innate immune responses in Atlantic salmon by chronic hypoxia-induced stress

Atlantic salmon post-smolts were exposed to either chronic hypoxic (Hy) or normal oxygen (No) conditions in seawater tanks for 58 days, mimicking conditions typical of sea cages for farmed salmon at some periods of the year. By day 29 head kidney macrophages were isolated and subjected to in vitro poly I:C stimulation to simulate viral infection, and samples were collected over 48 h. By day 58 fish were subjected to in vivo stimulation using poly I:C or a Vibrio water-based vaccine to simulate viral or bacterial infection, respectively. The fish were monitored for stress responses and expression of several pro-inflammatory genes in head kidney and intestinal tissue up to five days post-injection. Stress load was monitored by plasma cortisol estimation at days 29 and 58, and on days 1, 2, 3 and 5 post-injection in the in vivo trial. Hy exposure resulted in elevated plasma cortisol levels on day 29 compared to No, while on day 58 cortisol levels were higher in the control group. Additionally, both poly I:C and the Vibrio vaccine gave significantly increased cortisol levels one day post-injection compared to PBS treated controls, irrespective of previous oxygen exposure. In vitro stimulation of macrophages with poly I:C revealed higher IFNα mRNA levels at 6, 12 and 24 h and for Mx at 12 and 24 h post-stimulation, for both No and Hy individuals. Moreover, IFNα levels were higher in No than in Hy individuals at all time points, and a similar difference was seen in Mx at 48 h. In vivo stimulation with poly I:C elicited strong elevation of the IL-1β, IFNγ, Mx and IP10 mRNA transcripts in head kidney, while TNFα1 and IFNα were found unaffected. The Vibrio vaccine elicited a strong up regulation of IL-1β, IFNγ and IP10 mRNA, whereas Mx, TNFα1 and IFNα appeared unchanged. Significant differences in expression between different oxygen exposure groups were found for all genes and both stimuli. The overall trend suggests that long-term hypoxia either reduces or delays the expression of these genes in head kidney. Expression of IFNγ and Mx in intestinal tissues also showed a strong up regulation of the genes following poly I:C stimulation, and also here the overall trend suggests that chronic hypoxia results in a lower or delayed expression of the measured genes.In summary, our results indicate that chronic hypoxia modulates the expression of important immune related genes putatively altering the immune response. As the effect is present in isolated macrophages as well as head kidney and intestinal tissue the modulation appears to be affecting local as well as systemic responses.

The lifespan extension effects of resveratrol are conserved in the honey bee and may be driven by a mechanism related to caloric restriction

Our interest in healthy aging and in evolutionarily conserved mechanisms of lifespan extension prompted us to investigate whether features of age-related decline in the honey bee could be attenuated with resveratrol. Resveratrol is regarded as a caloric restriction mimetic known to extend lifespan in some but not all model species. The current, prevailing view is that resveratrol works largely by activating signaling pathways. It has also been suggested that resveratrol may act as an antioxidant and confer protection against nervous system impairment and oxidative stress. To test whether honey bee lifespan, learning performance, and food perception could be altered by resveratrol, we supplemented the diets of honey bees and measured lifespan, olfactory learning, and gustatory responsiveness to sucrose. Furthermore, to test the effects of resveratrol under metabolic challenge, we used hyperoxic environments to generate oxidative stress. Under normal oxygen conditions, two resveratrol treatments-30 and 130 μM-lengthened average lifespan in wild-type honey bees by 38% and 33%, respectively. Both resveratrol treatments also lengthened maximum and median lifespan. In contrast, hyperoxic stress abolished the resveratrol life-extension response. Furthermore, resveratrol did not affect learning performance, but did alter gustation. Honey bees that were not fed resveratrol exhibited greater responsiveness to sugar, while those supplemented with resveratrol were less responsive to sugar. We also discovered that individuals fed a high dose of resveratrol-compared to controls-ingested fewer quantities of food under ad libitum feeding conditions.