WorldWideScience

Sample records for cell hypoxia

  1. Maternal sickle cell trait and fetal hypoxia.

    Science.gov (United States)

    Manzar, S

    2000-01-01

    Patients with sickle cell trait (SCT) usually run a benign course. But they may develop vaso-occlusive crisis, which may lead to hypoxia. During these episodes, pregnant women with SCT may effect the developing fetus. This report describes an interesting finding of subtle degree of fetal hypoxia associated with maternal SCT. Twenty mothers with SCT were compared with 20 controls for the amount of circulating nucleated red blood cells (NRBC) and marker of fetal hypoxia at birth. Elevated number of circulating NRBC were noted in the cord blood of neonates born to mother with SCT as compared with controls, suggesting evidence of intrauterine fetal hypoxia. A larger prospective study is needed to elaborate further on this association.

  2. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  3. The radiation response of cells recovering after chronic hypoxia

    International Nuclear Information System (INIS)

    Kwok, T.T.; Sutherland, R.M.

    1989-01-01

    Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment

  4. Response of immortalized murine cementoblast cells to hypoxia in vitro.

    Science.gov (United States)

    Wu, Yeke; Han, Xianglong; Guo, Yongwen; Wu, Hao; Ren, Jing; Li, Jingyu; Ai, Dongqing; Wang, Lufei; Bai, Ding

    2013-11-01

    The aim of the study was to investigate the impact of hypoxia on proliferation, apoptosis and mineralization of cementoblast-like cells (OCCM-30) in vitro. The effects of different periods of hypoxia (2% O2) on proliferation, apoptosis, cementoblastic potential and root cementum resorption capability of OCCM-30 were evaluated, by using MTT, flow cytometry, alkaline phosphatase (ALP) activity assay, reverse transcription-polymerase chain reaction measurement, enzyme-linked immunosorbent assay and mineralization nodule formation assay. OCCM-30 viability was significantly inhibited by hypoxia while the apoptosis ratio was enhanced in a time-dependent manner; hypoxia inducible factor-1α and vascular endothelial growth factor mRNA were induced by hypoxia in different manners; temporary hypoxia (osteocalcin, bone sialoprotein, osteopontin and osteoprotegerin. In addition, hypoxia affected mineralized nodule formation of OCCM-30 in a time-dependent fashion; moreover, root cementum resorption function was also induced by hypoxia, manifested by increased receptor activator of nuclear factor kappa B ligand mRNA and protein expression. Temporary exposure of OCCM-30 to hypoxia inhibited proliferation, promoted apoptosis and mineralization, while longer duration of hypoxia could inhibit the cementoblast function. The findings may provide theoretical basis for developing novel therapeutics to prevent root resorption during orthodontic treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Sevoflurane suppresses hypoxia-induced growth and metastasis of lung cancer cells via inhibiting hypoxia-inducible factor-1α.

    Science.gov (United States)

    Liang, Hua; Yang, Cheng Xiang; Zhang, Bin; Wang, Han Bing; Liu, Hong Zhen; Lai, Xiao Hong; Liao, Mei Juan; Zhang, Tao

    2015-12-01

    Hypoxia promotes the progression of lung cancer cells. Unfortunately, anesthetic technique might aggravate hypoxia of lung cancer cells. Sevoflurane is a commonly used anesthetic. Its effect on hypoxia-induced aggressiveness of lung cancer cells remains unknown. The aim of the study is to investigate the effects of sevoflurane on hypoxia-induced growth and metastasis of lung cancer cells. As hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in mediating the adaptation and tolerance of cancer cells under hypoxic microenvironment, the role of HIF-1α in the effect of sevoflurane on hypoxia-induced growth and metastasis has also been elucidated. A549 cells were treated with normoxia, hypoxia, co-treatment of sevoflurane and hypoxia, and dimethyloxaloylglycine (DMOG, a HIF-1α agonist) for 4 h, respectively. MTT assay and colony formation assay were used to evaluate cell growth. Transwell assay was performed to detect invasion and migration ability. The protein level of HIF-1α, X-linked inhibitor of apoptosis protein (XIAP), survivin, fascin, heparanase (HPA), and p38 MAPK were determined by Western blotting. Hypoxia enhanced proliferation and metastatic potential of cells. Sevoflurane could suppress hypoxia-induced growth and metastasis ability of cells. Furthermore, HIF-1α, XIAP, survivin, fascin and HPA were down-regulated significantly by the co-treatment of sevoflurane and hypoxia as compared to hypoxia treatment. DMOG abolished the inhibiting effects of sevoflurane on hypoxia-induced growth and metastasis ability of cells. In addition, sevoflurane partly reversed the increase of p38 MAPK activity that was induced by hypoxia. Sevoflurane could suppress hypoxia-induced growth and metastasis of lung cancer cells, which might be associated with modulating HIF-1α and its down-stream genes. Moreover, p38 MAPK signaling pathway was involved in the regulation of HIF-1α by sevoflurane.

  6. Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

    African Journals Online (AJOL)

    induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

  7. Glycolysis determines dichotomous regulation of T cell subsets in hypoxia

    Science.gov (United States)

    Xu, Yang; Zhang, Ming; Savoldo, Barbara; Metelitsa, Leonid S.; Rodgers, John; Yustein, Jason T.; Neilson, Joel R.

    2016-01-01

    Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (TN and TCM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (TEM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in TN and TCM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate–rich elements in the 3′-UTR region of HIF1A mRNA in glycolytically inactive TN and TCM. Conversely, active glycolysis with decreased GAPDH availability in TEM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance TEM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue. PMID:27294526

  8. Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Bettina Schaible

    Full Text Available Pseudomonas aeruginosa (P. aeruginosa is an opportunistic pathogen commonly associated with lung and wound infections. Hypoxia is a frequent feature of the microenvironment of infected tissues which induces the expression of genes associated with innate immunity and inflammation in host cells primarily through the activation of the hypoxia-inducible factor (HIF and Nuclear factor kappaB (NF-κB pathways which are regulated by oxygen-dependent prolyl-hydroxylases. Hypoxia also affects virulence and antibiotic resistance in bacterial pathogens. However, less is known about the impact of hypoxia on host-pathogen interactions such as bacterial adhesion and infection. In the current study, we demonstrate that hypoxia decreases the internalization of P. aeruginosa into cultured epithelial cells resulting in decreased host cell death. This response can also be elicited by the hydroxylase inhibitor Dimethyloxallyl Glycine (DMOG. Reducing HIF-2α expression or Rho kinase activity diminished the effects of hypoxia on P. aeruginosa infection. Furthermore, in an in vivo pneumonia infection model, application of DMOG 48 h before infection with P. aeruginosa significantly reduced mortality. Thus, hypoxia reduces P. aeruginosa internalization into epithelial cells and pharmacologic manipulation of the host pathways involved may represent new therapeutic targets in the treatment of P. aeruginosa infection.

  9. Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function

    Science.gov (United States)

    McNamee, Eóin N.; Johnson, Darlynn Korns; Homann, Dirk

    2014-01-01

    Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies. PMID:22961658

  10. Hypoxia and the Presence of Human Vascular Endothelial Cells Affect Prostate Cancer Cell Invasion and Metabolism

    Directory of Open Access Journals (Sweden)

    Ellen Ackerstaff

    2007-12-01

    Full Text Available Tumor progression and metastasis are influenced by hypoxia, as well as by interactions between cancer cells and components of the stroma, such as endothelial cells. Here, we have used a magnetic resonance (MRcompatible invasion assay to further understand the effects of hypoxia on human prostate cancer cell invasion and metabolism in the presence and absence of human umbilical vein endothelial cells (HUVECs. Additionally, we compared endogenous activities of selected proteases related to invasion in PC-3 cells and HUVECs, profiled gene expression of PC-3 cells by microarray, evaluated cell proliferation of PC-3 cells and HUVECs by flow cytometry, under hypoxic and oxygenated conditions. The invasion of less-invasive DU-145 cells was not affected by either hypoxia or the presence of HUVECs. However, hypoxia significantly decreased the invasion of PC-3 cells. This hypoxia-induced decrease was attenuated by the presence of HUVECs, whereas under oxygenated conditions, HUVECs did not alter the invasion of PC-3 cells. Cell metabolism changed distinctly with hypoxia and invasion. The endogenous activity of selected extracellular proteases, although altered by hypoxia, did not fully explain the hypoxia-induced changes in invasion. Gene expression profiling indicated that hypoxia affects multiple cellular functions and pathways.

  11. Effects of hypoxia on human cancer cell line chemosensitivity

    Science.gov (United States)

    2013-01-01

    Background Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia. Methods A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Results Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Conclusions A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia. PMID:23829203

  12. Nitric Oxide And Hypoxia Response In Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Estefanía Caballano Infantes

    2015-08-01

    Full Text Available The expansion of pluripotent cells (ESCs and iPSCs under conditions that maintain their pluripotency is necessary to implement a cell therapy program. Previously, we have described that low nitric oxide (NO donor diethylenetriamine/nitric oxide adduct (DETA-NO added to the culture medium, promote the expansion of these cell types. The molecular mechanisms are not yet known. We present evidences that ESC and iPSCs in normoxia in presence of low NO triggers a similar response to hypoxia, thus maintaining the pluripotency. We have studied the stability of HIF-1α (Hypoxia Inducible Factor in presence of low NO. Because of the close relationship between hypoxia, metabolism, mitochondrial function and pluripotency we have analyzed by q RT-PCR the expression of genes involved in the glucose metabolism such as: HK2, LDHA and PDK1; besides other HIF-1α target gene. We further analyzed the expression of genes involved in mitochondrial biogenesis such as PGC1α, TFAM and NRF1 and we have observed that low NO maintains the same pattern of expression that in hypoxia. The study of the mitochondrial membrane potential using Mito-Tracker dye showed that NO decrease the mitochondrial function. We will analyze other metabolic parameters, to determinate if low NO regulates mitochondrial function and mimics Hypoxia Response. The knowledge of the role of NO in the Hypoxia Response and the mechanism that helps to maintain self-renewal in pluripotent cells in normoxia, can help to the design of culture media where NO could be optimal for stem cell expansion in the performance of future cell therapies.

  13. Glycogen synthesis is induced in hypoxia by the hypoxia-inducible factor and promotes cancer cell survival

    Directory of Open Access Journals (Sweden)

    Joffrey ePelletier

    2012-02-01

    Full Text Available The hypoxia-inducible factor 1 (HIF-1, in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1, were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these hypoxia-preconditioned cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO2 acts as an alarm that prepares the cells to face subsequent nutrient depletion and to survive.

  14. Hypoxia-ischemia and retinal ganglion cell damage

    Directory of Open Access Journals (Sweden)

    Charanjit Kaur

    2008-08-01

    Full Text Available Charanjit Kaur1, Wallace S Foulds2, Eng-Ang Ling11Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2Singapore Eye Research Institute, SingaporeAbstract: Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1α and its target genes such as vascular endothelial growth factor (VEGF and nitric oxide synthase (NOS. Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca2+ which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.Keywords: retinal hypoxia, retinal ganglion cells, glutamate receptors, neuronal injury, retina

  15. Cell-line dependent effects of hypoxia prior to irradiation in squamous cell carcinoma lines

    Directory of Open Access Journals (Sweden)

    Franziska Hauth

    2017-08-01

    Conclusion: We herein report a key role of ATM in the cellular fitness of cells exposed to prolonged moderate hypoxia prior to irradiation. While DNA damage response post-irradiation seem to be mainly driven by non-homologous end joining repair pathway in these conditions, our data suggest an important role for ATM kinase in hypoxia-driven modification of radiation response.

  16. Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells.

    Science.gov (United States)

    Jeon, Daun; Park, Heon Joo; Kim, Hong Seok

    2018-01-01

    Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial-mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Time-lapse microscopy of lung endothelial cells under hypoxia

    Science.gov (United States)

    Mehrvar, Shima; Ghanian, Zahra; Kondouri, Ganesh; Camara, Amadou S.; Ranji, Mahsa

    2017-02-01

    Objective: This study utilizes fluorescence microscopy to assess the effect of the oxygen tension on the production of reactive oxygen species (ROS) in mitochondria of fetal pulmonary artery endothelial cells (FPAECs). Introduction: Hypoxia is a severe oxygen stress, which mostly causes irreversible injury in lung cells. However, in some studies, it is reported that hypoxia decreases the severity of injuries. In this study, ROS production level was examined in hypoxic FPAECs treated with pentachlorophenol (PCP, uncoupler). This work was accomplished by monitoring and quantifying the changes in the level of the produced ROS in hypoxic cells before and after PCP treatment. Materials and methods: The dynamic of the mitochondrial ROS production in two groups of FPAECs was measured over time using time-lapse microscopy. For the first group, cells were incubated in 3% hypoxic condition for 2 hours and then continuously were exposed to hypoxic condition for imaging as well. For the second group, cells were incubated in normal oxygen condition. Time lapse images of the cells loaded with Mito-SOX (ROS indicator) were acquired, and the red fluorescence intensity profile of the cells was calculated. Changes in the level of the fluorescence intensity profile while they are treated with PCP indicates the dynamics of the ROS level. Results: The intensity profiles of the PCP-treated cells in the first group showed 47% lower ROS production rate than the PCP-treated cells in the second group. Conclusion: Time lapse microscopy revealed that hypoxic cells have lower ROS generation while treated with PCP. Therefore, this result suggests that hypoxia decreased electron transport chain activity in uncoupled chain.

  18. PKA activity exacerbates hypoxia-induced ROS formation and hypoxic injury in PC-12 cells.

    Science.gov (United States)

    Gozal, Evelyne; Metz, Cynthia J; Dematteis, Maurice; Sachleben, Leroy R; Schurr, Avital; Rane, Madhavi J

    2017-09-05

    Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death. Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O 2 ). Hypoxia, at 24h 0.1% O 2 , induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types. N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression. We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. The usability of a 15-gene hypoxia classifier as a universal hypoxia profile in various cancer cell types.

    Science.gov (United States)

    Sørensen, Brita Singers; Knudsen, Anders; Wittrup, Catja Foged; Nielsen, Steffen; Aggerholm-Pedersen, Ninna; Busk, Morten; Horsman, Michael; Høyer, Morten; Bouchelouche, Pierre Nourdine; Overgaard, Jens; Alsner, Jan

    2015-09-01

    A 15-gene hypoxia profile has previously demonstrated to have both prognostic and predictive impact for hypoxic modification in squamous cell carcinoma of the head and neck. This gene expression profile may also have a prognostic value in other histological cancer types, and could potentially have a function as a universal hypoxia profile. The hypoxia induced upregulation of the included genes, and the validity of the previously used reference genes was established in this study, in a range of different cell lines representing carcinomas of the prostate, colon, and esophagus. Eleven adenocarcinoma and one squamous cell lines: Six colon carcinomas (HTC8, HT29, LS174T, SW116, SW948 and T48), 3 prostate carcinomas (LNCaP, DU-145 and PC-3) and 3 esophagus carcinoma cell lines (OE19, OE21 and OE33) were cultured under normoxic or hypoxic conditions (0% O2) for 24hours. Total RNA was extracted and gene expression levels measured by qPCR. For each tissue type, individual reference genes were selected and applied in the normalization of the relative expression levels. In all three tissue types, individual, optimal, reference genes were selected. In the analysis of the hypoxia induced genes, both the original reference genes and the new selected reference genes were used. There was no significant difference in the obtained data. The gene expression analysis demonstrated cell line specific differences in the hypoxia response of the 15 genes, with BNIP3 not being upregulated at hypoxic conditions in 3 out of 6 colon cancer cell lines, and ALDOA in OE21 and FAM162A and SLC2A1 in SW116 only showing limited hypoxia induction. Furthermore, in the esophagus cell lines, the normoxic and hypoxic expression levels of LOX and BNIP3 were below the detection limit in OE19 and OE33, respectively. However, a combined analysis of the 15 genes in both adenocarcinoma cell lines and squamous carcinoma cell lines demonstrated a very consistent expression pattern in hypoxic induced gene

  20. Impact of Hypoxia on the Metastatic Potential of Human Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Dai Yao; Bae, Kyungmi; Siemann, Dietmar W.

    2011-01-01

    Purpose: Intratumoral hypoxia is known to be associated with radioresistance and metastasis. The present study examined the effect of acute and chronic hypoxia on the metastatic potential of prostate cancer PC-3, DU145, and LNCaP cells. Methods and Materials: Cell proliferation and clonogenicity were tested by MTT assay and colony formation assay, respectively. 'Wound-healing' and Matrigel-based chamber assays were used to monitor cell motility and invasion. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was tested by Western blot, and HIF-1-target gene expression was detected by real-time polymerase chain reaction. Secretion of matrix metalloproteinases (MMPs) was determined by gelatin zymography. Results: When PC-3 cells were exposed to 1% oxygen (hypoxia) for various periods of time, chronic hypoxia (≥24 h) decreased cell proliferation and induced cell death. In contrast, prostate cancer cells exposed to acute hypoxia (≤6 h) displayed increased motility, clonogenic survival, and invasive capacity. At the molecular level, both hypoxia and anoxia transiently stabilized HIF-1α. Exposure to hypoxia also induced the early expression of MMP-2, an invasiveness-related gene. Treatment with the HIF-1 inhibitor YC-1 attenuated the acute hypoxia-induced migration, invasion, and MMP-2 activity. Conclusions: The length of oxygen deprivation strongly affected the functional behavior of all three prostate cancer cell lines. Acute hypoxia in particular was found to promote a more aggressive metastatic phenotype.

  1. Effect of microenviroment hypoxia on glioma cells radiosensitivity through cancer stem cell pathway

    International Nuclear Information System (INIS)

    Tong Liumei; Feng Libo; Lu Xueguan; Chen Liesong; Guo Xinwei; Tian Ye

    2010-01-01

    Objective: To investigate the effect of microenviroment hypoxia on glioma cells radiosensitivity through cancer stem pathway, and to explore the related mechanism. Methods: Glioma cell lines SHG44 and U251 were cultured in normoxia (20% O 2 ) or continuous hypoxia (1% O 2 ) for 12 and 24 h. The fraction of glioma cells with positive expression of CD133 was assayed by flow cytometry. The radiosensitivity of glioma cells was determined by clonogenic cell assay. Western blotting was used to investigate the expressions of HIF-1 α and its downstream gene Notch 1. Results: The fraction of glioma cells with positive expression of CD133 was higher after hypoxic culture for 12 and 24 h than that of the corresponding cells cultured in normoxia. Compared to the cells cultured in normoxia, SF 2 (survival fraction at 2 Gy) were enhanced significantly in SHG44 and U251 cells cultured in hypoxia for 12 and 24 h. The OER (oxygen-enhancement ratio) of SHG44 cells in hypoxia for 12 and 24 h was 1.54 and 1.38, respectively. The OER of U251 cells was 1.44 and 1.23, respectively. The radiosensitivity of these two cell line was decreased in hypoxia. The protein expressions of HIF-1 α and Notch 1 genes were elevated more significantly for cells cultured in hypoxia for 12 and 24 h than for those in normoxia. Conclusions: Microenviroment hypoxia could increase the radioresistance of glioma cells through enrichment of cancer stem cells, and HIF-1 α-Notch 1 signal pathway may play an important role in this process. (authors)

  2. Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

    Science.gov (United States)

    Yermolaieva, Olena; Xu, Rong; Schinstock, Carrie; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2004-02-01

    Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of methionine in proteins involving the enzyme peptide methionine sulfoxide reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

  3. Comparative and Experimental Studies on the Genes Altered by Chronic Hypoxia in Human Brain Microendothelial Cells

    Directory of Open Access Journals (Sweden)

    Eugenia Mata-Greenwood

    2017-05-01

    Full Text Available Background : Hypoxia inducible factor 1 alpha (HIF1A is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia.Results : We examined the effect of chronic hypoxia (3 days; 0.5% oxygen on human brain micro endothelial cells (HBMEC viability and apoptosis. Hypoxia caused a significant reduction in cell viability and an increase in apoptosis. Next, we examined chronic hypoxia associated changes in transcriptome and genome-wide promoter methylation. The data obtained was compared with 16 other microarray studies on chronic hypoxia. Nine genes were altered in response to chronic hypoxia in all 17 studies. Interestingly, HIF1A was not altered with chronic hypoxia in any of the studies. Furthermore, we compared our data to three other studies that identified HIF-responsive genes by various approaches. Only two genes were found to be HIF dependent. We silenced each of these 9 genes using CRISPR/Cas9 system. Downregulation of EGLN3 significantly increased the cell death under chronic hypoxia, whereas downregulation of ERO1L, ENO2, adrenomedullin, and spag4 reduced the cell death under hypoxia.Conclusions : We provide a core group of genes that regulates cellular acclimatization under chronic hypoxic stress, and most of them are HIF independent.

  4. Propofol Reversed Hypoxia-Induced Docetaxel Resistance in Prostate Cancer Cells by Preventing Epithelial-Mesenchymal Transition by Inhibiting Hypoxia-Inducible Factor 1α.

    Science.gov (United States)

    Qian, Jiang; Shen, Sheliang; Chen, Wei; Chen, Nianping

    2018-01-01

    Prostate cancer is the second most frequently diagnosed cancer worldwide. Hypoxia-induced epithelial-mesenchymal transition (EMT), driven by hypoxia-inducible factor 1 α (HIF-1 α ), is involved in cancer progression and metastasis. The present study was designed to explore the role of propofol in hypoxia-induced resistance of prostate cancer cells to docetaxel. We used the Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine incorporation assay to measure cell viability and cell proliferation, respectively, in prostate cancer cell lines. Then, we detected HIF-1 α , E-cadherin, and vimentin expression using western blotting. Propofol reversed the hypoxia-induced docetaxel resistance in the prostate cancer cell lines. Propofol not only decreased hypoxia-induced HIF-1 α expression, but also reversed hypoxia-induced EMT by suppressing HIF-1 α . Furthermore, small interfering RNA-mediated silencing of HIF-1 α reversed the hypoxia-induced docetaxel resistance, although there was little change in docetaxel sensitivity between the hypoxia group and propofol group. The induction of hypoxia did not affect E-cadherin and vimentin expression, and under the siRNA knockdown conditions, the effects of propofol were obviated. These data support a role for propofol in regulating EMT in prostate cancer cells. Taken together, our findings demonstrate that propofol plays an important role in hypoxia-induced docetaxel sensitivity and EMT in prostate cancer cells and that it is a potential drug for overcoming drug resistance in prostate cancer cells via HIF-1 α suppression.

  5. Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway.

    Science.gov (United States)

    Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng; Qiao, Yong

    2016-01-01

    Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia-reperfusion injury. © 2015 by the Society for Experimental Biology and Medicine.

  6. Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia.

  7. The usability of a 15-gene hypoxia classifier as a universal hypoxia profile in various cancer cell types

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Knudsen, Anders Bisgård; Wittrup, Catja Foged

    2015-01-01

    , and esophagus. MATERIALS AND METHODS: Eleven adenocarcinoma and one squamous cell lines: Six colon carcinomas (HTC8, HT29, LS174T, SW116, SW948 and T48), 3 prostate carcinomas (LNCaP, DU-145 and PC-3) and 3 esophagus carcinoma cell lines (OE19, OE21 and OE33) were cultured under normoxic or hypoxic conditions...... genes, with BNIP3 not being upregulated at hypoxic conditions in 3 out of 6 colon cancer cell lines, and ALDOA in OE21 and FAM162A and SLC2A1 in SW116 only showing limited hypoxia induction. Furthermore, in the esophagus cell lines, the normoxic and hypoxic expression levels of LOX and BNIP3 were below...... the tissue type dependency of hypoxia induced genes included in a 15-gene hypoxic profile in carcinoma cell lines from prostate, colon, and esophagus cancer, and demonstrated that in vitro, with minor fluctuations, the genes in the hypoxic profile are hypoxia inducible, and the hypoxia profile may...

  8. Gene Expression Programs in Response to Hypoxia: Cell Type Specificity and Prognostic Significance in Human Cancers.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available BACKGROUND: Inadequate oxygen (hypoxia triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF, plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases. METHODS AND FINDINGS: We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use. CONCLUSIONS: The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis

  9. Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.

    Science.gov (United States)

    Olszewska-Pazdrak, Barbara; Hein, Travis W; Olszewska, Paulina; Carney, Darrell H

    2009-05-01

    Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

  10. Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack

    Energy Technology Data Exchange (ETDEWEB)

    Scherbakov, Alexander M., E-mail: alex.scherbakov@gmail.com [Laboratory of Clinical Biochemistry, Institute of Clinical Oncology, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow 115478 (Russian Federation); Stefanova, Lidia B.; Sorokin, Danila V.; Semina, Svetlana E. [Laboratory of Molecular Endocrinology, Institute of Carcinogenesis, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow 115478 (Russian Federation); Berstein, Lev M. [Laboratory of Oncoendocrinology, N.N. Petrov Research Institute of Oncology, St. Petersburg 197758 (Russian Federation); Krasil’nikov, Mikhail A. [Laboratory of Molecular Endocrinology, Institute of Carcinogenesis, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow 115478 (Russian Federation)

    2013-12-10

    The tolerance of cancer cells to hypoxia depends on the combination of different factors – from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial–mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O{sub 2} atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK – the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling. Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well

  11. HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

    International Nuclear Information System (INIS)

    Liu, Junye; Zhang, Jing; Wang, Xiaowu; Li, Yan; Chen, Yongbin; Li, Kangchu; Zhang, Jian; Yao, Libo; Guo, Guozhen

    2010-01-01

    Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

  12. Propofol Reversed Hypoxia-Induced Docetaxel Resistance in Prostate Cancer Cells by Preventing Epithelial–Mesenchymal Transition by Inhibiting Hypoxia-Inducible Factor 1α

    Directory of Open Access Journals (Sweden)

    Jiang Qian

    2018-01-01

    Full Text Available Prostate cancer is the second most frequently diagnosed cancer worldwide. Hypoxia-induced epithelial–mesenchymal transition (EMT, driven by hypoxia-inducible factor 1α (HIF-1α, is involved in cancer progression and metastasis. The present study was designed to explore the role of propofol in hypoxia-induced resistance of prostate cancer cells to docetaxel. We used the Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine incorporation assay to measure cell viability and cell proliferation, respectively, in prostate cancer cell lines. Then, we detected HIF-1α, E-cadherin, and vimentin expression using western blotting. Propofol reversed the hypoxia-induced docetaxel resistance in the prostate cancer cell lines. Propofol not only decreased hypoxia-induced HIF-1α expression, but also reversed hypoxia-induced EMT by suppressing HIF-1α. Furthermore, small interfering RNA–mediated silencing of HIF-1α reversed the hypoxia-induced docetaxel resistance, although there was little change in docetaxel sensitivity between the hypoxia group and propofol group. The induction of hypoxia did not affect E-cadherin and vimentin expression, and under the siRNA knockdown conditions, the effects of propofol were obviated. These data support a role for propofol in regulating EMT in prostate cancer cells. Taken together, our findings demonstrate that propofol plays an important role in hypoxia-induced docetaxel sensitivity and EMT in prostate cancer cells and that it is a potential drug for overcoming drug resistance in prostate cancer cells via HIF-1α suppression.

  13. Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage

    Directory of Open Access Journals (Sweden)

    Kumar Anil

    2010-10-01

    Full Text Available Abstract Background The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6 plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis. Results We have shown that RGCs exposed to hypoxia (1% or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM, revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6. Conclusion Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.

  14. HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

    Directory of Open Access Journals (Sweden)

    Chen-Ye Shi

    2013-08-01

    Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

  15. Delayed cell death signaling in traumatized central nervous system: hypoxia.

    Science.gov (United States)

    Chu, Danielle; Qiu, JingXin; Grafe, Marjorie; Fabian, Roderick; Kent, Thomas A; Rassin, David; Nesic, Olivera; Werrbach-Perez, Karin; Perez-Polo, Regino

    2002-02-01

    There are two different ways for cells to die: necrosis and apoptosis. Cell death has traditionally been described as necrotic or apoptotic based on morphological criteria. There are controversy about the respective roles of apoptosis and necrosis in cell death resulting from trauma to the central nervous system (CNS). An evaluation of work published since 1997 in which electron microscopy was applied to ascertain the role of apoptosis and necrosis in: spinal cord injury, stroke, and hypoxia/ischemia (H/I) showed evidence for necrosis and apoptosis based on DNA degradation, presence of histones in cytoplasm, and morphological evidence in spinal cord. In the aftermath of stroke, many of the biochemical markers for apoptosis were present but the morphological determinations suggested that necrosis is the major source of post-traumatic cell death. This was not the case in H/I where both biochemical assays and the morphological studies gave more consistent results in a manner similar to the spinal cord injury studies. After H/I, major factors affecting cell death outcomes are DNA damage and repair processes, expression of bcl-like gene products and inflammation-triggered cytokine production.

  16. Protective effects of bellidifolin in hypoxia-induced in pheochromocytoma cells (PC12) and underlying mechanisms.

    Science.gov (United States)

    Zhao, Zhi-Ying; Gao, Yang-Yang; Gao, Li; Zhang, Ming; Wang, He; Zhang, Chun-Hong

    2017-01-01

    Bellidifolin, a xanthone compound derived from plants of Gentiana species, is known to exert a variety of pharmacological activities including anti-oxidation, anti-inflammatory and antitumor actions as well as a protective effect on cerebral ischemic nerve injury. The aim of this study was to examine the protective effects of bellidifolin on nerve injury produced by hypoxia and possible underlying mechanisms using pheochromocytoma cells (PC12). Data showed that the viability of PC12 cells subjected to hypoxia resulted in a significant decrease; however; pretreatment with certain concentrations of bellidifolin (20 or 40 μmol/L) prior to hypoxia significantly increased the survival rate. The results of immunohistochemical staining analysis revealed that there were no marked alterations in the expression of pERK protein between all bellidifolin groups while the expression of p-p38MAPK protein was significantly enhanced by hypoxia. Pretreatment with different concentrations of bellidifolin followed by hypoxia significantly decreased the expression of p-p38MAPK protein. The results of western blot analysis showed that hypoxia induced the expression of the MAPK signaling pathway downstream of the key apoptosis factor caspase-3. Compared to hypoxia, the expression of caspase-3 in the presence of belliidifolin was significantly lower. Data suggest that bellidifolin may contribute to the protective effects associated with nerve injury initiated by hypoxia by mechanisms related to inhibition of cell apoptosis independent of the ERK pathway, but may involve blockade of p38MAPK signaling pathway activation and downstream caspase-3 expression.

  17. Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk; Lee, YoungJoo, E-mail: yjlee@sejong.ac.kr

    2016-02-12

    Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERα protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.

  18. Hypoxia enhances proliferation through increase of colony formation rate with chondrogenic potential in primary synovial mesenchymal stem cells.

    Science.gov (United States)

    Ohara, Toshiyuki; Muneta, Takeshi; Nakagawa, Yusuke; Matsukura, Yu; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

    2016-01-01

    Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Use of primary MSCs is the preferable because these cells are safer than cells passaged several times in terms of probability of chromosome abnormalities. The effect of hypoxia on the proliferation of MSCs is controversial and remains unknown in primary synovial MSCs. Primary synovial MSCs were cultured at normoxia or hypoxia, and colony number, cell number, surface epitopes, mitochondria activity, TEM finding, and chondrogenic potential were analyzed. To investigate the effect of hypoxia on attachment of synovial MSCs, cells were cultured at hypoxia for the first 3 days, then cultured at normoxia. To investigate the effect of hypoxia on proliferation, cells were also cultured at hypoxia for the last 11 days. Hypoxia increased colony number and cell number per dish in primary synovial MSCs. Hypoxia did not affect cell number per colony, surface epitopes, mitochondria activity, TEM finding or chondrogenic potential. Hypoxia for the first 3 days did not alter colony number per dish or cell number per dish, while hypoxia for the last 11 days increased. Hypoxia enhanced proliferation through increase of colony formation rate with chondrogenic potential in primary synovial MSCs.

  19. Chronic hypoxia promotes pulmonary artery endothelial cell proliferation through H2O2-induced 5-lipoxygenase.

    Directory of Open Access Journals (Sweden)

    Kristi M Porter

    Full Text Available Pulmonary Hypertension (PH is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5. While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC were cultured under normoxic (21% O2 or hypoxic (1% O2 conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2 release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.

  20. Association of hypoxia inducible factor 1 (HIF-1 and paraoxonase enzyme in lung cancer cell lines

    Directory of Open Access Journals (Sweden)

    Nedim Karagenç

    2015-09-01

    Full Text Available Objective: In this study, it was aimed to investigate that PON2, in hypoxia and normoxia in relation to HIF-1α transcription factor that has a role in tumour angiogenesis. Hypoxia inducible factor 1 alpha (HIF-1α is a transcription factor which is sensitive to hypoxia, causes initiation of angiogenesis and metastasis by providing transcription of numerous genes. As well as hypoxia several inflammatory agents such as lipopolysaccharide (LPS can regulate the expression of HIF-1α as well as PON2. Oxidative stress is known to have role in cancer. Paraoxonase 2 which is one of the members of paraoxonase family serves as intracellular anti-oxidant. Methods: H1299, A549 ve PC14 non-small cell lung carcinoma cell lines used in this study. Cells were cultured under hypoxia and normoxia conditions with LPS stimulation. HIF-1α and PON2 mRNA expression levels measured by real-time PCR. Western blot studies were performed for protein expression. Results: In this study, it was observed that LPS treatment stimulates HIF-1α expression which increases PON2 expression in NSCLC cell line in under hxpoxia conditions. Conclusion: This study shows that PON2 is regulated by HIF-1α in hypoxia and inflammation. The relationship between hypoxia and inflammation and oxidative status of cells requires further studies.

  1. Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mahendran Botlagunta

    2011-03-01

    Full Text Available DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

  2. Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting.

    Science.gov (United States)

    Centanin, Lázaro; Dekanty, Andrés; Romero, Nuria; Irisarri, Maximiliano; Gorr, Thomas A; Wappner, Pablo

    2008-04-01

    Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.

  3. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

    Science.gov (United States)

    Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-01-01

    Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

  4. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    Directory of Open Access Journals (Sweden)

    Lorena Olivares-González

    Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  5. The critical role of ERK in death resistance and invasiveness of hypoxia-selected glioblastoma cells

    International Nuclear Information System (INIS)

    Kim, Jee-Youn; Kim, Yong-Jun; Lee, Sun; Park, Jae-Hoon

    2009-01-01

    The rapid growth of tumor parenchyma leads to chronic hypoxia that can result in the selection of cancer cells with a more aggressive behavior and death-resistant potential to survive and proliferate. Thus, identifying the key molecules and molecular mechanisms responsible for the phenotypic changes associated with chronic hypoxia has valuable implications for the development of a therapeutic modality. The aim of this study was to identify the molecular basis of the phenotypic changes triggered by chronic repeated hypoxia. Hypoxia-resistant T98G (HRT98G) cells were selected by repeated exposure to hypoxia and reoxygenation. Cell death rate was determined by the trypan blue exclusion method and protein expression levels were examined by western blot analysis. The invasive phenotype of the tumor cells was determined by the Matrigel invasion assay. Immunohistochemistry was performed to analyze the expression of proteins in the brain tumor samples. The Student T-test and Pearson Chi-Square test was used for statistical analyses. We demonstrate that chronic repeated hypoxic exposures cause T98G cells to survive low oxygen tension. As compared with parent cells, hypoxia-selected T98G cells not only express higher levels of anti-apoptotic proteins such as Bcl-2, Bcl-X L , and phosphorylated ERK, but they also have a more invasive potential in Matrigel invasion chambers. Activation or suppression of ERK pathways with a specific activator or inhibitor, respectively, indicates that ERK is a key molecule responsible for death resistance under hypoxic conditions and a more invasive phenotype. Finally, we show that the activation of ERK is more prominent in malignant glioblastomas exposed to hypoxia than in low grade astrocytic glial tumors. Our study suggests that activation of ERK plays a pivotal role in death resistance under chronic hypoxia and phenotypic changes related to the invasive phenotype of HRT98G cells compared to parent cells

  6. Hypoxia impairs primordial germ cell migration in zebrafish (Danio rerio embryos.

    Directory of Open Access Journals (Sweden)

    Kwok Hong Lo

    Full Text Available As a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear.In the current study, the effect of hypoxia on primordial germ cell (PGC migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1, an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration.This study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1.

  7. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

    International Nuclear Information System (INIS)

    Khan, Shaheen; Liu Shengxi; Stoner, Matthew; Safe, Stephen

    2007-01-01

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERα crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1α or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFκB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide

  8. Effect of hypoxia on thallium kinetics in cultured chick myocardial cells

    International Nuclear Information System (INIS)

    Friedman, B.J.; Beihn, R.; Friedman, J.P.

    1987-01-01

    To assess the effect of hypoxia on cellular thallium-201 ( 201 Tl) uptake and washout independent of coronary flow, we studied thallium kinetics during normoxia and hypoxia in cultured chick ventricular cells. Monolayers of contracting ventricular cells grown on coverslips were placed in a chamber and perfused to asymptote with media containing 201 Tl. Perfusates were equilibrated with 5% CO 2 -95% air or 5% CO 2 -95% nitrogen for normoxia and hypoxia, respectively. Washout thallium kinetics were then observed during perfusion with unlabeled media. Twenty paired experiments were performed, randomly alternating the sequence of normoxia and hypoxia. Pharmacokinetics for thallium were determined by computer using standard formulae. Thallium uptake and washout were best described by assuming that intracellular thallium was contained within a single compartment. Cellular thallium uptake, as well as transfer rate constants for thallium uptake and for thallium washout during normoxia and hypoxia, were compared using paired t-tests. During normoxia and hypoxia, respectively, thallium uptake was 22 +/- 7% and 19 +/- 7% of asymptote (p less than 0.01); the compartmental rate constant for uptake by the cell was 0.16 +/- 0.07 min-1 and 0.15 +/- 0.06 min-1 (N.S.); and the transfer rate constant for washout from the cell was 0.26 +/- 0.06 min-1 and 0.23 +/- 0.05 min-1 (p less than 0.01). We conclude that there was a small (14%) decrease in thallium uptake during hypoxia. The rate of thallium uptake and washout was slightly less during hypoxia, although only the rate of washout was significantly less. These data show that cellular accumulation of thallium and the rate of washout of thallium were minimally decreased by hypoxia independent of blood flow

  9. Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, You-Kyoung [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Sae-Gwang; Choi, Il-Whan [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Soo-Woong [Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Sang Min [Department of Internal Medicine, Division of Hematology/Oncology, Busan Paik Hospital, Inje University, Busan 614-735 (Korea, Republic of); Choi, Inhak, E-mail: miccih@inje.ac.kr [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)

    2015-04-03

    Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

  10. Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension.

    Science.gov (United States)

    Maston, Levi D; Jones, David T; Giermakowska, Wieslawa; Howard, Tamara A; Cannon, Judy L; Wang, Wei; Wei, Yongyi; Xuan, Weimin; Resta, Thomas C; Gonzalez Bosc, Laura V

    2017-05-01

    Inflammation is a prominent pathological feature in pulmonary arterial hypertension, as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4 + T cells are required for initiating and maintaining inflammation, suggesting that these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Our objective was to test the hypothesis that CD4 + T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 wk) in wild-type mice and recombination-activating gene 1 knockout mice (RAG1 -/- , lacking mature T and B cells). Separate sets of mice were adoptively transferred with CD4 + , CD8 + , or T helper 17 cells before normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. RAG1 -/- mice had diminished right ventricular systolic pressure and arterial remodeling compared with wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4 + but not CD8 + T cells restored the hypertensive phenotype in RAG1 -/- mice. Interestingly, RAG1 -/- mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4 + cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. We conclude that T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension. Copyright © 2017 the American Physiological Society.

  11. Moderate hypoxia induces β-cell dysfunction with HIF-1-independent gene expression changes.

    Directory of Open Access Journals (Sweden)

    Yoshifumi Sato

    Full Text Available Pancreatic β-cell failure is central to the development and progression of type 2 diabetes. We recently demonstrated that β-cells become hypoxic under high glucose conditions due to increased oxygen consumption and that the pancreatic islets of diabetic mice but not those of control mice are moderately hypoxic. However, the impact of moderate hypoxia on β-cell number and function is unknown. In the present study, moderate hypoxia induced a hypoxic response in MIN6 cells, as evidenced by increased levels of HIF-1α protein and target genes. Under these conditions, a selective downregulation of Mafa, Pdx1, Slc2a2, Ndufa5, Kcnj11, Ins1, Wfs1, Foxa2, and Neurod1, which play important roles in β-cells, was also observed in both MIN6 cells and isolated pancreatic islets. Consistent with the altered expression of these genes, abnormal insulin secretion was detected in hypoxic MIN6 cells. Most of the hypoxia-induced gene downregulation in MIN6 cells was not affected by the suppression of HIF-1α, suggesting a HIF-1-independent mechanism. Moderate hypoxia also induced apoptosis in MIN6 cells. These results suggest that hypoxia is a novel stressor of β-cells and that hypoxic stress may play a role in the deterioration of β-cell function.

  12. Dormancy of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia.

    Directory of Open Access Journals (Sweden)

    Hiroko Endo

    Full Text Available A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death in vitro and a decreased number of dormant cells in vivo. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor-depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer.

  13. Cell physiology regulation by hypoxia inducible factor-1: Targeting oxygen-related nanomachineries of hypoxic cells.

    Science.gov (United States)

    Eskandani, Morteza; Vandghanooni, Somayeh; Barar, Jaleh; Nazemiyeh, Hossein; Omidi, Yadollah

    2017-06-01

    Any dysfunctionality in maintaining the oxygen homeostasis by mammalian cells may elicit hypoxia/anoxia, which results in inescapable oxidative stress and possible subsequent detrimental impacts on certain cells/tissues with high demands to oxygen molecules. The ischemic damage in turn can trigger initiation of a number of diseases including organs ischemia, metabolic disorders, inflammatory diseases, different types of malignancies, and alteration in wound healing process. Thus, full comprehension of molecular mechanism(s) and cellular physiology of the oxygen homeostasis is the cornerstone of the mammalian cells metabolism, energetic pathways and health and disease conditions. An imbalance in oxygen content within the cellular microenvironment activates a cascade of molecular events that are often compensated, otherwise pathologic condition occurs through a complexed network of biomolecules. Hypoxia inducible factor-1 (HIF-1) plays a key transcriptional role in the adaptation of cell physiology in relation with the oxygen content within a cell. In this current study, we provide a comprehensive review on the molecular mechanisms of oxygen sensing and homeostasis and the impacts of HIF-1 in hypoxic/anoxic conditions. Moreover, different molecular and biochemical responses of the cells to the surrounding environment are discussed in details. Finally, modern technological approaches for targeting the hypoxia related proteins are articulated. Copyright © 2017. Published by Elsevier B.V.

  14. Carvedilol abrogates hypoxia-induced oxidative stress and neuroinflammation in microglial BV2 cells.

    Science.gov (United States)

    Gao, Xiujuan; Wu, Bin; Fu, Zhijian; Zhang, Zongwang; Xu, Guangjun

    2017-11-05

    Microglia initially undergo rapid activation in response to injury and stressful stimuli, such as hypoxia. Oxidative stress and the inflammatory response play critical roles in hypoxic-ischemic brain injury. Carvedilol is a β-blocker used to treat high blood pressure and heart failure. In this study, we investigated whether carvedilol had a protective effect against hypoxia-induced oxidative stress and inflammation in microglial BV2 cells. Our results indicate that hypoxic exposure significantly reduced mean cell viability of BV2 microglia, which was significantly restored by carvedilol (10 and 50μM). In addition, carvedilol treatment significantly inhibited the hypoxia-induced increase in reactive oxygen species (ROS) and 4-hydroxy-2-nonenal (4-HNE). Administration of carvedilol significantly inhibited expression of IL-1β, TNF-α, and IL-6 at both the mRNA and protein levels. Mechanistically, we found that hypoxia significantly increased phosphorylation of IKK, IκBα, and NF-κB p65. However, treatment with carvedilol inhibited phosphorylation of these molecules. Notably, hypoxia resulted in a significant nuclear translocation of NF-κB p65, which was inhibited by administration of carvedilol. Luciferase reporter assay results demonstrate that treatment with carvedilol inhibited the hypoxia-induced increase in NF-κB binding activity. These data suggest that carvedilol may be of potential use as a novel therapy against hypoxia or ischemia. Copyright © 2017. Published by Elsevier B.V.

  15. Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells.

    Science.gov (United States)

    Zeitouni, Nathalie E; Dersch, Petra; Naim, Hassan Y; von Köckritz-Blickwede, Maren

    2016-01-01

    Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins.

  16. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    International Nuclear Information System (INIS)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-01-01

    Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

  17. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  18. HDAC6 deacetylase activity is required for hypoxia-induced invadopodia formation and cell invasion.

    Directory of Open Access Journals (Sweden)

    Dominique Arsenault

    Full Text Available Despite significant progress in the cancer field, tumor cell invasion and metastasis remain a major clinical challenge. Cell invasion across tissue boundaries depends largely on extracellular matrix degradation, which can be initiated by formation of actin-rich cell structures specialized in matrix degradation called invadopodia. Although the hypoxic microenvironment within solid tumors has been increasingly recognized as an important driver of local invasion and metastasis, little is known about how hypoxia influences invadopodia biogenesis. Here, we show that histone deacetylase 6 (HDAC6, a cytoplasmic member of the histone deacetylase family, is a novel modulator of hypoxia-induced invadopodia formation. Hypoxia was found to enhance HDAC6 tubulin deacetylase activity through activation of the EGFR pathway. Activated HDAC6, in turn, triggered Smad3 phosphorylation resulting in nuclear accumulation. Inhibition of HDAC6 activity or knockdown of the protein inhibited both hypoxia-induced Smad3 activation and invadopodia formation. Our data provide evidence that hypoxia influences invadopodia formation in a biphasic manner, which involves the activation of HDAC6 deacetylase activity by EGFR, resulting in enhanced Smad phosphorylation and nuclear accumulation. The identification of HDAC6 as a key participant of hypoxia-induced cell invasion may have important therapeutic implications for the treatment of metastasis in cancer patients.

  19. Hypoxia promotes etoposide (VP-16) resistance in neuroblastoma CHP126 cells.

    Science.gov (United States)

    Wang, Duoduo; Zhu, Qionghua; Zhang, Xiayan; Zhang, Lei; He, Qiaojun; Yang, Bo

    2010-01-01

    Hypoxia is widespread in solid tumors as a consequence of poorly structured tumor-derived neovasculature, which is recognized to play a role in the resistance of cancer cells to chemotherapy. Etoposide (VP-16), a drug commonly used in chemotherapy, leads to enhanced accumulation of cell populations in G2/M phase and increases levels of apoptosis as a topoisomerase II inhibitor. We evaluated the effects of hypoxia on the response of the neuroblastoma cell line CHP126 to VP-16, in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance of this clinically conventional anti-cancer agent, with an insight to determining potential indications in neuroblastoma therapy. In this study, physiological hypoxia was shown to attenuate G2/M arrest and apoptosis induced in CHP126 cells by VP-16. It suppressed drug-related Cdk1 activity with a less elevation of regulator proteins such as cyclin B1, Cdk7 and reduced caspase activation and PARP cleavage compared to the efficiency observed in normoxic condition, which were significantly relative with hypoxia-driven inhibition of p53 and p-ERK1/2 activation. These results clearly demonstrated that hypoxia had a protective effect against VP-16-induced cytotoxicity, which is likely to provide a further therapeutic knowledge in neuroblastomas.

  20. The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

    International Nuclear Information System (INIS)

    Chen, J.K.; Hu, L.J.; Kong, E.L.; Lamborn, K.R.; Deen, D.F.

    2003-01-01

    Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

  1. The Effect of Perinatal Hypoxia on Red Blood Cell Morphology in Newborns

    OpenAIRE

    S. A. Perepelitsa; V. A. Sergunova; O. E. Gudkova

    2017-01-01

    Aim. To study the red blood cell (RBC) morphology in newborn infants with a history of perinatal hypoxia using the atomic-force microscopy. Material and methods. The state of RBC membranes of 10 newborns with a history of perinatal hypoxia was studied. All infants were born with low Apgar scoring; the following resuscitative measures were carried out at birth: tracheal intubation, mechanical ventilation (MV). The study group newborns were transferred from the delivery room to the ICU, where M...

  2. Mathematical modelling of phenotypic plasticity and conversion to a stem-cell state under hypoxia

    Science.gov (United States)

    Dhawan, Andrew; Madani Tonekaboni, Seyed Ali; Taube, Joseph H.; Hu, Stephen; Sphyris, Nathalie; Mani, Sendurai A.; Kohandel, Mohammad

    2016-02-01

    Hypoxia, or oxygen deficiency, is known to be associated with breast tumour progression, resistance to conventional therapies and poor clinical prognosis. The epithelial-mesenchymal transition (EMT) is a process that confers invasive and migratory capabilities as well as stem cell properties to carcinoma cells thus promoting metastatic progression. In this work, we examined the impact of hypoxia on EMT-associated cancer stem cell (CSC) properties, by culturing transformed human mammary epithelial cells under normoxic and hypoxic conditions, and applying in silico mathematical modelling to simulate the impact of hypoxia on the acquisition of CSC attributes and the transitions between differentiated and stem-like states. Our results indicate that both the heterogeneity and the plasticity of the transformed cell population are enhanced by exposure to hypoxia, resulting in a shift towards a more stem-like population with increased EMT features. Our findings are further reinforced by gene expression analyses demonstrating the upregulation of EMT-related genes, as well as genes associated with therapy resistance, in hypoxic cells compared to normoxic counterparts. In conclusion, we demonstrate that mathematical modelling can be used to simulate the role of hypoxia as a key contributor to the plasticity and heterogeneity of transformed human mammary epithelial cells.

  3. The Effect of Perinatal Hypoxia on Red Blood Cell Morphology in Newborns

    Directory of Open Access Journals (Sweden)

    S. A. Perepelitsa

    2017-01-01

    Full Text Available Aim. To study the red blood cell (RBC morphology in newborn infants with a history of perinatal hypoxia using the atomic-force microscopy. Material and methods. The state of RBC membranes of 10 newborns with a history of perinatal hypoxia was studied. All infants were born with low Apgar scoring; the following resuscitative measures were carried out at birth: tracheal intubation, mechanical ventilation (MV. The study group newborns were transferred from the delivery room to the ICU, where MV was started. To obtain images of normal red blood cells in the field of the atomic force microscope (AFM, 14 full-term newborns delivered after a favorable course of pregnancy and normal term labor were enrolled in a reference group. Results. Discocytes and planocytes comprised 36% of the total red blood cell count in the residual umbilical cord blood of newborns with a history of perinatal hypoxia; there was a decreased amount of normal RBC forms, thus demonstrating an unfavorable effect of hypoxia on newborn's RBC membrane. Poikilocytosis was typical for infants exposed to perinatal hypoxia; transitional forms of RBCs (stomatocytes and echynocytes were visualized. Stomatocytosis and echynocytosis were typical for 80% of newborns. Stomatocytosis persisted in full-term newborns exposed to hypoxia complicated with aspiration of neonatal meconium. The analysis of RBC membrane nanostructure demonstrated that the first-order height (h1 experienced the greatest alterations at birth in newborns with perinatal hypoxia; it was 4.2 times as much as the similar parameter in healthy newborns. Estimations of second-order height (h2 parameter values demonstrated a two-fold increase showing that the spectrin matrix also changed under the effect of hypoxia. The third order value (h3 was significantly higher in newborns with perinatal hypoxia, than that in healthy infants. Therefore, perinatal hypoxia causes antenatal complete damage of nanostructures of RBC membranes

  4. Exogenous wild type p53 gene affects radiosensitivity of human lung adenocarcinoma cell line under hypoxia

    International Nuclear Information System (INIS)

    Wang Jianhua; Wang Feng; Liu Yongping; Zhang Yaping; Ni Yan; Li Shirong

    2008-01-01

    Objective: To evaluate the effect of exogenous wild type p53 (wtp53) gene on radiosensitivity of human lung adenocarcinoma cell line under hypoxia. Methods: Human lung adenocarcinoma cell line A549 was transfected with adenovirus carrying recombinant exogenous wtp53. Four irradiation groups were studied: normal cell (Group A), wtp53 transfected cell (Group B), normal cell under hypoxia (Group C) and wtp53 transfected cell under hypoxia(Group D). Cells were irradiated with 9 MeV electron beams. Cellular survival fraction was analyzed. Multi-target single-hit model was used to plot the survival curve. D 0 , D q , oxygen enhancement ratio (OER), sensitizing enhancement ratio (SER) and other parameters were used to evaluate the effects of wtp53 gene on radiosensitivity of A549. The cell apoptotic rate of each group was examined by flow cytometry. Results: OER was 1.75 and 0.81 before and after wtp53 transfection. SER was 1.77 in oxic circumstance and 3.84 under hypoxia. The cell apoptotic rate of Group A and B was lower than Group C and D (F=7.92, P=0.048), with Group A lower than B and Group C lower than D (F=82.50, P=0.001). But Group B and D were similar(t=2.04, P=0.111). Conclusions: Hypoxia can increase the radiation resistance of lung adenocarcinoma cell line A549. The wtp53 can promote apoptosis and improve tumor radiosensitivity, especially under hypoxia. (authors)

  5. Comparative SAGE analysis of the response to hypoxia in human pulmonary and aortic endothelial cells.

    Science.gov (United States)

    Peters, D G; Ning, W; Chu, T J; Li, C J; Choi, A M K

    2006-07-12

    We utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human pulmonary artery endothelial cells (HPAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HPAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same-passage cells cultured under standard (5% CO2-95% air) conditions. Hierarchical clustering of significant hypoxia-responsive genes identified temporal changes in the expressions of a number of well-described gene families including those encoding proteins involved in thrombosis, stress response, apoptosis, angiogenesis, and cell proliferation. These experiments build on previously published data describing the transcriptomic response of human aortic endothelial cells (HAECs) obtained from the same donor and cultured under identical conditions, and we have thus taken advantage of the immortality of SAGE data to make direct comparisons between these two data sets. This approach revealed comprehensive information relating to the similarities and differences at the level of mRNA expression between HAECs and HPAECs. For example, we found differences in the cell type-specific response to hypoxia among genes encoding cytoskeletal factors, including paxillin, and proteins involved in metabolic energy production, the response to oxidative stress, and vasoreactivity (e.g., endothelin-1). These efforts contribute to the expanding collection of publicly available SAGE data and provide a foundation on which to base further efforts to understand the characteristics of the vascular response to hypoxia in the pulmonary circulation relative to systemic vasculature.

  6. Physiological Hypoxia Enhances Stemness Preservation, Proliferation, and Bidifferentiation of Induced Hepatic Stem Cells

    Directory of Open Access Journals (Sweden)

    Xiaosong Zhi

    2018-01-01

    Full Text Available Induced hepatic stem cells (iHepSCs have great potential as donors for liver cell therapy due to their self-renewal and bipotential differentiation properties. However, the efficiency of bidifferentiation and repopulation efficiency of iHepSCs is relatively low. Recent evidence shows that physiological hypoxia, a vital factor within stem cell “niche” microenvironment, plays key roles in regulating tissue stem cell biological behaviors including proliferation and differentiation. In this study, we found that physiological hypoxia (10% O2 enhanced the stemness properties and promoted the proliferation ability of iHepSCs by accelerating G1/S transition via p53-p21 signaling pathway. In addition, short-term hypoxia preconditioning improved the efficiency of hepatic differentiation of iHepSCs, and long-term hypoxia promoted cholangiocytic differentiation but inhibited hepatic differentiation of iHepSCs. These results demonstrated the potential effects of hypoxia on stemness preservation, proliferation, and bidifferentiation of iHepSCs and promising perspective to explore appropriate culture conditions for therapeutic stem cells.

  7. Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Law, Alice Y.S. [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong); Wong, Chris K.C., E-mail: ckcwong@hkbu.edu.hk [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong)

    2010-02-01

    Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1{alpha} dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

  8. Bcl-2 family members make different contributions to cell death in hypoxia and/or hyperoxia in rat cerebral cortex.

    Science.gov (United States)

    Hu, Xiaoming; Qiu, Jingxin; Grafe, Marjorie R; Rea, Harriett C; Rassin, David K; Perez-Polo, J Regino

    2003-11-01

    Hypoxic brain injury during fetal or neonatal development leads to damaged immature neurons and can result in cognitive or behavioral dysfunction. Hyperoxia therapy (treatment with oxygen) is commonly applied to infants with signs of perinatal hypoxia-anoxia. Both hypoxia and hyperoxia have been shown to result in apoptosis in the brains of rats in several animal models. One determinant of cellular commitment to cell death is the differential expression of the Bcl-2 family of proteins in response to trauma. Here, we characterize cell death and the expression of Bcl-2 homologous proteins in 7-day-old neonatal rat cerebral cortex after hypoxia (5% O(2) for 40 min) and/or hyperoxia (>95% O(2) for 2 h after hypoxia). The expression of Bcl-2 and Bcl-X(L), two anti-apoptotic proteins, decreased at 24 h after hypoxia. Bcl-X(L) increased after either hyperoxia or hypoxia+hyperoxia. We did not detect significant changes in the cytoplasmic levels of pro-apoptotic protein Bax after any of these three treatments. Using cell death ELISA and DNA FragEL assays, we observed increased cell death at 24h after hypoxia, hyperoxia or hypoxia+hyperoxia treatments. At 24 h after either hypoxia, hyperoxia or hypoxia+hyperoxia, caspase 3 activity also increased significantly. Our results suggest that both hypoxia and hyperoxia alone can induce cell death. The Bcl-2 --> cytochrome c --> caspase 3 pathway played a role in hypoxia-induced cell death, while other pathways may be involved in hyperoxia-induced cell death.

  9. Zeaxanthin Inhibits Hypoxia-Induced VEGF Secretion by RPE Cells through Decreased Protein Levels of Hypoxia-Inducible Factors-1α

    Directory of Open Access Journals (Sweden)

    Richard Rosen

    2015-01-01

    Full Text Available Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization.

  10. Antioxidant Mechanism of Rutin on Hypoxia-Induced Pulmonary Arterial Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Qian Li

    2014-11-01

    Full Text Available Reactive oxygen species (ROS are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4 in pulmonary artery endothelial cells (PAECs. Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α. Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC, a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.

  11. Antioxidant mechanism of Rutin on hypoxia-induced pulmonary arterial cell proliferation.

    Science.gov (United States)

    Li, Qian; Qiu, Yanli; Mao, Min; Lv, Jinying; Zhang, Lixin; Li, Shuzhen; Li, Xia; Zheng, Xiaodong

    2014-11-18

    Reactive oxygen species (ROS) are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC) proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4) in pulmonary artery endothelial cells (PAECs). Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α). Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC), a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.

  12. RNAi Knockdown of Hypoxia-Inducible Factor-1α Decreased the Proliferation, Migration, and Invasion of Hypoxic Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Chen, ChengShi; Liu, Rong; Wang, JianHua; Yan, ZhiPing; Qian, Sheng; Zhang, Wei

    2015-04-01

    The obstruction of hepatic arterial blood flow results in tumor tissue hypoxia and elevated expression of hypoxia-inducible factor-1alpha (HIF-1α). Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells under hypoxia. RNA interference knockdown of HIF-1α was achieved by HIF-1α-directed lentiviral shRNA, in a rat HCC cell line cultured under hypoxia condition for varying length of times. The expression levels of HIF-1α and vascular endothelial growth factor were examined using reverse transcription polymerase chain reaction and western blot analyses. Cell proliferation, migration, and invasion were measured by cell viability, transwell migration, and invasion assays, respectively. Inhibition of HIF-1α expression by shRNA suppressed vascular endothelial growth factor mRNA and protein levels under both normoxia and hypoxia. It also suppressed cell migration and invasion, which were enhanced under hypoxic conditions. RNAi knockdown of HIF-1α further suppressed hypoxia-mediated inhibition of the cell proliferation. These data suggest that shRNA of HIF-1α could antagonize the hypoxia-mediated increase in hepatic cancer cell migration and invasion, and synergize with hypoxia to inhibit the cell proliferation in HCC cells.

  13. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

    International Nuclear Information System (INIS)

    Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine

    2006-01-01

    To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours

  14. Notch1 is required for hypoxia-induced proliferation, invasion and chemoresistance of T-cell acute lymphoblastic leukemia cells

    Directory of Open Access Journals (Sweden)

    Zou Jie

    2013-01-01

    Full Text Available Abstract Background Notch1 is a potent regulator known to play an oncogenic role in many malignancies including T-cell acute lymphoblastic leukemia (T-ALL. Tumor hypoxia and increased hypoxia-inducible factor-1α (HIF-1α activity can act as major stimuli for tumor aggressiveness and progression. Although hypoxia-mediated activation of the Notch1 pathway plays an important role in tumor cell survival and invasiveness, the interaction between HIF-1α and Notch1 has not yet been identified in T-ALL. This study was designed to investigate whether hypoxia activates Notch1 signalling through HIF-1α stabilization and to determine the contribution of hypoxia and HIF-1α to proliferation, invasion and chemoresistance in T-ALL. Methods T-ALL cell lines (Jurkat, Sup-T1 transfected with HIF-1α or Notch1 small interference RNA (siRNA were incubated in normoxic or hypoxic conditions. Their potential for proliferation and invasion was measured by WST-8 and transwell assays. Flow cytometry was used to detect apoptosis and assess cell cycle regulation. Expression and regulation of components of the HIF-1α and Notch1 pathways and of genes related to proliferation, invasion and apoptosis were assessed by quantitative real-time PCR or Western blot. Results Hypoxia potentiated Notch1 signalling via stabilization and activation of the transcription factor HIF-1α. Hypoxia/HIF-1α-activated Notch1 signalling altered expression of cell cycle regulatory proteins and accelerated cell proliferation. Hypoxia-induced Notch1 activation increased the expression of matrix metalloproteinase-2 (MMP2 and MMP9, which increased invasiveness. Of greater clinical significance, knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with losing the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression. Conclusions Notch1 signalling is required for hypoxia/HIF-1α-induced proliferation

  15. Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Wooyoung [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of); Bazer, Fuller W. [Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A& M University, College Station, TX (United States); Song, Gwonhwa, E-mail: ghsong@korea.ac.kr [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Jinyoung, E-mail: jinyoungkim@dankook.ac.kr [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of)

    2016-01-08

    The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

  16. Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

    International Nuclear Information System (INIS)

    Jeong, Wooyoung; Bazer, Fuller W.; Song, Gwonhwa; Kim, Jinyoung

    2016-01-01

    The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

  17. Hypoxia-induced acidification causes mitoxantrone resistance not mediated by drug transporters in human breast cancer cells

    NARCIS (Netherlands)

    Greijer, A.E.; Jong, M.C. de; Scheffer, G.L.; Shvarts, A.; Diest, P.J. van; Wall, E. van der

    2005-01-01

    Hypoxia has clinically been associated with resistance to chemotherapy. The aim of this study was to investigate whether hypoxia induces resistance to doxorubicin and mitoxantrone, two common drugs in cancer treatment, in MCF-7 breast cancer cells, and SW1573 non-small lung cancer cells. In

  18. Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia.

    Science.gov (United States)

    Li, Rong; Wang, Li-Zhao; Du, Jun-Hui; Zhao, Lei; Yao, Yang

    2018-01-01

    To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells (RMECs) under hypoxia stimulation. The murine RMECs were primarily cultured and randomly divided into three groups: hypoxia group (cultured in 1% O 2 environment), hypoxia+autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine (3-MA) for 4h followed by incubation in 1% O 2 ] and control group (cultured under normoxic condition). The state of autophagy in RMECs was examined by assaying the turnover of light chain 3B (LC3BB) and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy (TEM) and by counting the number of GFP+ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-mTOR were also assayed by Western blotting. Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-II/I and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP+ puncta were also increased under hypoxia. However, pre-treatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-mTOR level was decreased in cells exposed to hypoxia. In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.

  19. Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis.

    Science.gov (United States)

    Lan, Ying-Wei; Choo, Kong-Bung; Chen, Chuan-Mu; Hung, Tsai-Hsien; Chen, Young-Bin; Hsieh, Chung-Hsing; Kuo, Han-Pin; Chong, Kowit-Yu

    2015-05-20

    Idiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs. Hypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration. The expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the

  20. Regulation of matrix stiffness on the epithelial-mesenchymal transition of breast cancer cells under hypoxia environment

    Science.gov (United States)

    Lv, Yonggang; Chen, Can; Zhao, Boyuan; Zhang, Xiaomei

    2017-06-01

    Substrate stiffness and hypoxia are associated with tumor development and progression, respectively. However, the synergy of them on the biological behavior of human breast cancer cell is still largely unknown. This study explored how substrate stiffness regulates the cell phenotype, viability, and epithelial-mesenchymal transition (EMT) of human breast cancer cells MCF-7 under hypoxia (1% O2). TRITC-phalloidin staining showed that MCF-7 cells transformed from round to irregular polygon with stiffness increase either in normoxia or hypoxia. While being accompanied with the upward tendency from a 0.5- to a 20-kPa substrate, the percentage of cell apoptosis was significantly higher in hypoxia than that in normoxia, especially on the 20-kPa substrate. Additionally, it was hypoxia, but not normoxia, that promoted the EMT of MCF-7 by upregulating hypoxia-inducible factor-1α (HIF-1α), vimentin, Snail 1, and matrix metalloproteinase 2 (MMP 2) and 9 (MMP 9), and downregulating E-cadherin simultaneously regardless of the change of substrate stiffness. In summary, this study discovered that hypoxia and stiffer substrate (20 kPa) could synergistically induce phenotype change, apoptosis, and EMT of MCF-7 cells. Results of this study have an important significance on further exploring the synergistic effect of stiffness and hypoxia on the EMT of breast cancer cells and its molecular mechanism.

  1. Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells.

    Science.gov (United States)

    Renteria, Lissette S; Raj, J Usha; Ibe, Basil O

    2010-12-01

    Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO(2)<40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO(2) ~100 torr) conditions. PAF binding was quantified in fmol/10(6) cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45-60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Telomere elongation protects heart and lung tissue cells from fatal damage in rats exposed to severe hypoxia.

    Science.gov (United States)

    Wang, Yaping; Zhao, Zhen; Zhu, Zhiyong; Li, Pingying; Li, Xiaolin; Xue, Xiaohong; Duo, Jie; Ma, Yingcai

    2018-02-17

    The effects of acute hypoxia at high altitude on the telomere length of the cells in the heart and lung tissues remain unclear. This study aimed to investigate the change in telomere length of rat heart and lung tissue cells in response to acute exposure to severe hypoxia and its role in hypoxia-induced damage to heart and lung tissues. Forty male Wistar rats (6-week old) were randomized into control group (n = 10) and hypoxia group (n = 30). Rats in control group were kept at an altitude of 1500 m, while rats in hypoxia group were exposed to simulated hypoxia with an altitude of 5000 m in a low-pressure oxygen chamber for 1, 3, and 7 days (n = 10). The left ventricular and right middle lobe tissues of each rat were collected for measurement of telomere length and reactive oxygen species (ROS) content, and the mRNA and protein levels of telomerase reverse transcriptase (TERT), hypoxia-inducible factor1α (HIF-1α), and hypoxia-inducible factor1α (HIF-2α). Increased exposure to hypoxia damaged rat heart and lung tissue cells and increased ROS production and telomere length. The mRNA and protein levels of TERT and HIF-1α were significantly higher in rats exposed to hypoxia and increased with prolonged exposure; mRNA and protein levels of HIF-2α increased only in rats exposed to hypoxia for 7 days. TERT was positively correlated with telomere length and the levels of HIF-1α but not HIF-2α. Acute exposure to severe hypoxia causes damage to heart and lung tissues due to the production of ROS but promotes telomere length and adaptive response by upregulating TERT and HIF-1α, which protect heart and lung tissue cells from fatal damage.

  3. Moderate hypoxia influences potassium outward currents in adipose-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Mayuri Prasad

    Full Text Available Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K+ channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K+ channel blocker tetraethylammonium (TEA was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K+ currents composed by Ca2+-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K+ channels.

  4. Functional and anatomical evidence of cerebral tissue hypoxia in young sickle cell anemia mice.

    Science.gov (United States)

    Cahill, Lindsay S; Gazdzinski, Lisa M; Tsui, Albert Ky; Zhou, Yu-Qing; Portnoy, Sharon; Liu, Elaine; Mazer, C David; Hare, Gregory Mt; Kassner, Andrea; Sled, John G

    2017-03-01

    Cerebral ischemia is a significant source of morbidity in children with sickle cell anemia; however, the mechanism of injury is poorly understood. Increased cerebral blood flow and low hemoglobin levels in children with sickle cell anemia are associated with increased stroke risk, suggesting that anemia-induced tissue hypoxia may be an important factor contributing to subsequent morbidity. To better understand the pathophysiology of brain injury, brain physiology and morphology were characterized in a transgenic mouse model, the Townes sickle cell model. Relative to age-matched controls, sickle cell anemia mice demonstrated: (1) decreased brain tissue pO 2 and increased expression of hypoxia signaling protein in the perivascular regions of the cerebral cortex; (2) elevated basal cerebral blood flow , consistent with adaptation to anemia-induced tissue hypoxia; (3) significant reduction in cerebrovascular blood flow reactivity to a hypercapnic challenge; (4) increased diameter of the carotid artery; and (5) significant volume changes in white and gray matter regions in the brain, as assessed by ex vivo magnetic resonance imaging. Collectively, these findings support the hypothesis that brain tissue hypoxia contributes to adaptive physiological and anatomic changes in Townes sickle cell mice. These findings may help define the pathophysiology for stroke in children with sickle cell anemia.

  5. Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Hong [Department of Ophthalmology, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China); The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China); Wu, Xinyi, E-mail: xywu8868@163.com [Department of Ophthalmology, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-{beta}. Black-Right-Pointing-Pointer Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. Black-Right-Pointing-Pointer Hypoxia inhibits Acanthamoeba-induced the activation of NF-{kappa}B and ERK1/2 in HCECs. Black-Right-Pointing-Pointer Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. Black-Right-Pointing-Pointer LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-{beta} (IFN-{beta}) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-{beta}. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-{kappa}B and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88

  6. Hypoxia and Human Genome Stability: Downregulation of BRCA2 Expression in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Daniele Fanale

    2013-01-01

    Full Text Available Previously, it has been reported that hypoxia causes increased mutagenesis and alteration in DNA repair mechanisms. In 2005, an interesting study showed that hypoxia-induced decreases in BRCA1 expression and the consequent suppression of homologous recombination may lead to genetic instability. However, nothing is yet known about the involvement of BRCA2 in hypoxic conditions in breast cancer. Initially, a cell proliferation assay allowed us to hypothesize that hypoxia could negatively regulate the breast cancer cell growth in short term in vitro studies. Subsequently, we analyzed gene expression in breast cancer cell lines exposed to hypoxic condition by microarray analysis. Interestingly, genes involved in DNA damage repair pathways such as mismatch repair, nucleotide excision repair, nonhomologous end-joining and homologous recombination repair were downregulated. In particular, we focused on the BRCA2 downregulation which was confirmed at mRNA and protein level. In addition, breast cancer cells were treated with dimethyloxalylglycine (DMOG, a cell-permeable inhibitor of both proline and asparaginyl hydroxylases able to induce HIF-1α stabilization in normoxia, providing results comparable to those previously described. These findings may provide new insights into the mechanisms underlying genetic instability mediated by hypoxia and BRCA involvement in sporadic breast cancers.

  7. Differential effect of hypoxia on human mesenchymal stem cell chondrogenesis and hypertrophy in hyaluronic acid hydrogels.

    Science.gov (United States)

    Zhu, Meiling; Feng, Qian; Bian, Liming

    2014-03-01

    Photocrosslinked hyaluronic acid (HA) hydrogels provide a conducive 3-D environment that supports the chondrogenesis of human mesenchymal stem cells (hMSCs). The HA macromer concentration in the hydrogels has a significant impact on the chondrogenesis of the encapsulated MSCs due to changes in the physical properties of the hydrogels. Meanwhile, hypoxia has been shown to promote MSC chondrogenesis and suppress subsequent hypertrophy. This study investigates the combinatorial effect of tuning HA macromer concentration (1.5-5%w/v) and hypoxia on MSC chondrogenesis and hypertrophy. To decouple the effect of HA concentration from that of crosslinking density, the HA hydrogel crosslinking density was adjusted by varying the extent of the reaction through the light exposure time while keeping the HA concentration constant (5%w/v at 5 or 15 min). It was found that hypoxia had no significant effect on the chondrogenesis and cartilaginous matrix synthesis of hMSCs under all hydrogel conditions. In contrast, the hypoxia-mediated positive or negative regulation of hMSC hypertrophy in HA hydrogels is dependent on the HA concentration but independent of the crosslinking density. Specifically, hypoxia significantly suppressed hMSC hypertrophy and neocartilage calcification in low HA concentration hydrogels, whereas hypoxia substantially enhanced hMSC hypertrophy, leading to elevated tissue calcification in high HA concentration hydrogels irrespective of their crosslinking density. In addition, at a constant high HA concentration, increasing hydrogel crosslinking density promoted hMSC hypertrophy and matrix calcification. To conclude, the findings from this study demonstrate that the effect of hypoxia on hMSC chondrogenesis and hypertrophy is differentially influenced by the encapsulating HA hydrogel properties. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  8. Hypoxia targeting therapy with prodrug specifically stabilized and activated in hypoxic tumor cells

    International Nuclear Information System (INIS)

    Kondoh, S.K.; Ueda, T.; Harada, H.; Hiraoka, M.; Akagi, K.

    2003-01-01

    Hypoxia fraction in tumors is associated with increased metastasis and poor survival in patients suffering from malignant tumors such as the head and neck, cervical or breast cancers. Hypoxia can be a direct cause of therapeutic resistance because some drugs and radiation require oxygen to be maximally cytotoxic. Recently we have reported a novel hypoxia targeting prodrug, TOP3, which is a fusion protein, composed of HIV TAT protein transduction domain, a part of HIF1 α ODD domain, and Procaspase-3. TOP3 can be transferred into every cell both in vitro and in vivo but becomes stable only in hypoxic cells, in which TOP3 is activated and induces apoptosis. The application of this fusion protein to a tumor-bearing mouse resulted in significant suppression of the tumor growth and even in reduction of the tumor mass without any obvious side effects. The administrations of TOP3 in combination with a low dose of X-ray showed an additive antitumor effect on pancreatic tumor cells. Furthermore, we show that the rodent model of ascites generated by malignant cells provides an excellent platform of testing hypoxia targeting drugs, since it comprises homogeneous fluid with tumor cells surviving and proliferating under hypoxic condition. TOP3 induced apoptosis of AH130, rat ascites hepatoma cells, in vitro only under hypoxic but not normoxic condition. Intraperitoneal administration of TOP3 prolonged life span of the rats with AH130 derived malignant ascites. Sixty percent of the treated rats were cured of ascites without recurrence for more than six months, in contrast all untreated rats died within 20 days after tumor cell inoculation. These results strongly suggest that TOP3 would provide a new strategy for hypoxia targeting therapy and that the combination of TOP3 with radiotherapy or chemotherapy may provide a new strategy for annihilating malignant tumors

  9. TMEM45A is essential for hypoxia-induced chemoresistance in breast and liver cancer cells

    International Nuclear Information System (INIS)

    Flamant, Lionel; Roegiers, Edith; Pierre, Michael; Hayez, Aurélie; Sterpin, Christiane; De Backer, Olivier; Arnould, Thierry; Poumay, Yves; Michiels, Carine

    2012-01-01

    Hypoxia is a common characteristic of solid tumors associated with reduced response to radio- and chemotherapy, therefore increasing the probability of tumor recurrence. The aim of this study was to identify new mechanisms responsible for hypoxia-induced resistance in breast cancer cells. MDA-MB-231 and HepG2 cells were incubated in the presence of taxol or etoposide respectively under normoxia and hypoxia and apoptosis was analysed. A whole transcriptome analysis was performed in order to identify genes whose expression profile was correlated with apoptosis. The effect of gene invalidation using siRNA was studied on drug-induced apoptosis. MDA-MB-231 cells incubated in the presence of taxol were protected from apoptosis and cell death by hypoxia. We demonstrated that TMEM45A expression was associated with taxol resistance. TMEM45A expression was increased both in MDA-MB-231 human breast cancer cells and in HepG2 human hepatoma cells in conditions where protection of cells against apoptosis induced by chemotherapeutic agents was observed, i.e. under hypoxia in the presence of taxol or etoposide. Moreover, this resistance was suppressed by siRNA-mediated silencing of TMEM45A. Kaplan Meier curve showed an association between high TMEM45A expression and poor prognostic in breast cancer patients. Finally, TMEM45 is highly expressed in normal differentiated keratinocytes both in vitro and in vivo, suggesting that this protein is involved in epithelial functions. Altogether, our results unravel a new mechanism for taxol and etoposide resistance mediated by TMEM45A. High levels of TMEM45A expression in tumors may be indicative of potential resistance to cancer therapy, making TMEM45A an interesting biomarker for resistance

  10. Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

    International Nuclear Information System (INIS)

    Pan, Hong; Wu, Xinyi

    2012-01-01

    Highlights: ► Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-β. ► Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. ► Hypoxia inhibits Acanthamoeba-induced the activation of NF-κB and ERK1/2 in HCECs. ► Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. ► LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion

  11. Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Michele Tanturli

    Full Text Available We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML cells and selects stem cells where BCR/Abl(protein is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1, by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2 and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening.

  12. Effects of hypoxia on osteogenic differentiation of rat bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Wang, Yating; Li, Juan; Wang, Yanmin; Lei, Lei; Jiang, Chunmiao; An, Shu; Zhan, Yuxiang; Cheng, Qian; Zhao, Zhihe; Wang, Jun; Jiang, Lingyong

    2012-03-01

    Bone reconstruction is essential in orthodontic treatment that caters to the correction of malocclusion by bone reconstruction. Mesenchymal stem cells (MSCs) have been demonstrated a great potency of osteogenesis. The aim of this study was to investigate the effect of hypoxia on the rat bone marrow MSCs (rBMSCs) in vitro during osteogenesis. In this study, we found that temporary exposure of rBMSCs after osteogenic induction for 7 days to hypoxia (2% oxygen) led to a marked decrease in ALPase activity and the expression of osteocalcin and Runt related transcription factor 2/core binding factor a1 (Runx2/Cbfa1). Meanwhile, we found that exposure to hypoxia led to an early and transient increase in the level of phosphorylated ERK1/2 but had no obvious effects on mitogen-activated protein kinase (p38 MAPK) level. Based on these results, we concluded that hypoxia could inhibit osteogenic differentiation of rBMSCs possibly through MEK-ERK 1/2, while p38 MAPK may not participate in this regulation. Further exploration into the mechanisms of hypoxia on osteogenesis would surely provide reliable evidence for clinical practice.

  13. [Study on proliferation and function of periodontal ligament fibroblasts and osteoblastic cells under hypoxia].

    Science.gov (United States)

    Kubota, M

    1989-12-01

    There have been many reports recognizing vascular changes on pressure side of periodontal tissues during orthodontic tooth movement. The vascular changes cause local hypoxia which seems to affect the phenotypes of periodontal tissue cells. In order to clarify the effect of hypoxia on proliferation and function of periodontal tissue cells, DNA content, alkaline phosphatase (ALP) activity and prostaglandin E2 (PGE2) production under a hypoxic condition in both periodontal ligament fibroblasts (PLF) and osteoblastic cells (MC3T3-E1 cells) were examined in vitro. PLF were cultured from human periodontium and identified by both morphologic characterization and presence of ALP. The results obtained were as follows: 1. Under 10% O2 condition, the activity of proliferation in PLF did not change but that of osteoblasts was inhibited. 2. ALP activity in PLF was stimulated but that of osteoblasts was inhibited under the hypoxic condition. 3. Production of PGE2 in osteoblasts increased after 7 days of hypoxia though that in PLF decreased. In addition, the enhancement of PGE2 production in osteoblasts was due to activation of both phospholipase A2 and PGE2-synthesizing enzymes. 4. From the orthodontic point of view, hypoxia on the pressure side may induce bone resorption by inhibiting mineralization activity of osteoblasts and enhancing production of PGE2 in osteoblasts.

  14. Hypoxia Pathway Proteins As Central Mediators of Metabolism in the Tumor Cells and Their Microenvironment

    Directory of Open Access Journals (Sweden)

    Sundary Sormendi

    2018-01-01

    Full Text Available Low oxygen tension or hypoxia is a determining factor in the course of many different processes in animals, including when tissue expansion and cellular metabolism result in high oxygen demands that exceed its supply. This is mainly happening when cells actively proliferate and the proliferating mass becomes distant from the blood vessels, such as in growing tumors. Metabolic alterations in response to hypoxia can be triggered in a direct manner, such as the switch from oxidative phosphorylation to glycolysis or inhibition of fatty acid desaturation. However, as the modulated action of hypoxia-inducible factors or the oxygen sensors (prolyl hydroxylase domain-containing enzymes can also lead to changes in enzyme expression, these metabolic changes can also be indirect. With this review, we want to summarize our current knowledge of the hypoxia-induced changes in metabolism during cancer development, how they are affected in the tumor cells and in the cells of the microenvironment, most prominently in immune cells.

  15. Neuronal death after perinatal cerebral hypoxia-ischemia: Focus on autophagy-mediated cell death.

    Science.gov (United States)

    Descloux, C; Ginet, V; Clarke, P G H; Puyal, J; Truttmann, A C

    2015-10-01

    Neonatal hypoxic-ischemic encephalopathy is a critical cerebral event occurring around birth with high mortality and neurological morbidity associated with long-term invalidating sequelae. In view of the great clinical importance of this condition and the lack of very efficacious neuroprotective strategies, it is urgent to better understand the different cell death mechanisms involved with the ultimate aim of developing new therapeutic approaches. The morphological features of three different cell death types can be observed in models of perinatal cerebral hypoxia-ischemia: necrotic, apoptotic and autophagic cell death. They may be combined in the same dying neuron. In the present review, we discuss the different cell death mechanisms involved in neonatal cerebral hypoxia-ischemia with a special focus on how autophagy may be involved in neuronal death, based: (1) on experimental models of perinatal hypoxia-ischemia and stroke, and (2) on the brains of human neonates who suffered from neonatal hypoxia-ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Effects of Hypoxia and Chitosan on Equine Umbilical Cord-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    D. J. Griffon

    2016-01-01

    Full Text Available Chitosan opens new perspectives in regenerative medicine as it enhances the properties of mesenchymal stem cells (MSCs through formation of spheroids. Hypoxia has also been proposed to enhance stemness and survival of MSCs after in vivo implantation. These characteristics are relevant to the development of an off-the-shelf source of allogenic cells for regenerative therapy of tendinopathies. Umbilical cord-derived MSCs (UCM-MSCs offer an abundant source of immature and immunoprivileged stem cells. In this study, equine UCM-MSCs (eqUCM-MSCs conditioned for 3 and 7 days on chitosan films at 5% oxygen were compared to eqUCM-MSCs under standard conditions. Equine UCM-MSCs formed spheroids on chitosan but yielded 72% less DNA than standard eqUCM-MSCs. Expression of Sox2, Oct4, and Nanog was 4 to 10 times greater in conditioned cells at day 7. Fluorescence-labeled cells cultured for 7 days under standard conditions or on chitosan films under hypoxia were compared in a bilateral patellar tendon defect model in rats. Fluorescence was present in all treated tendons, but the modulus of elasticity under tension was greater in tendons treated with conditioned cells. Chitosan and hypoxia affected cell yield but improved the stemness of eqUCM-MSCs and their contribution to the healing of tissues. Given the abundance of allogenic cells, these properties are highly relevant to clinical applications and outweigh the negative impact on cell proliferation.

  17. Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells

    Directory of Open Access Journals (Sweden)

    Tetsuro Yasui

    2017-06-01

    Full Text Available Human neural precursor cells (hNPCs derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

  18. The proliferation, apoptosis, invasion of endothelial-like epithelial ovarian cancer cells induced by hypoxia

    Directory of Open Access Journals (Sweden)

    Zhu Pengfei

    2010-09-01

    Full Text Available Abstract Background Epithelial ovarian cancer is one of the most malignant cancers in women because metastasis occurs in the most of patients by the time of diagnosis. Cancer cells have strong capacity to form angiogenesis or vasculogenic mimicry, which plays the major role in its malignant phenotype. Vasculogenic mimicry might contribute to the failure of the angiogenesis-targeted therapy strategies. Under the microenvironment of the tumor, hypoxia is the most common phenomena because of the vast energy and oxygen consuming. In the present study, the endothelial-like cells induced by hypoxia from SKOV-3 and ES-2 ovarian cancer cells were harvested to investigate the changes in their biological behaviors. Methods The endothelial-like cells from SKOV-3 and ES-2 cells were harvested by laser capture microdissection. The biological behaviors of the endothelial-like cells, including proliferation, cell cycle, apoptosis, invasion and telomerase activity were determined by MTT, FCM, Transwell chamber and TRAP-ELISA methods. HIF-1α is the most important factor for the behavior changes under hypoxic condition. Some other genes relative to biological behaviors are also changes following the changes of HIF-1α. In order to elucidate the underlying mechanisms for these changes by hypoxia, the relative genes expressions including HIF-1α, CyclinD1, Flk-1, VEGF, p53 and V-src were determined by real-time PCR. Results SKOV-3 and ES-2 cells were resistant to hypoxia by adoption of proliferation, apoptosis, differentiation and invasion. Combined with other studies, the more poorly cancer cells differentiate, the more strongly cells are resistant to hypoxia, the more possible to form vasculogenic mimicry. The changes in the expression of HIF-1α, and HIF-1α-dependent VEGF, Flk-1, Cyclin D1, and HIF-1α-independent p53 have been involved in this process. Conclusions HIF-1α took an important role in the behavioral changes of SKOV-3 and ES-2 cells by hypoxia. At

  19. Hypoxia increases the metastatic ability of breast cancer cells via upregulation of CXCR4

    LENUS (Irish Health Repository)

    Cronin, Patricia A

    2010-05-21

    Abstract Background Chemokine SDF1α and its unique receptor CXCR4 have been implicated in organ-specific metastases of many cancers including breast cancer. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. We hypothesized that hypoxia would upregulate CXCR4 expression and lead to increased chemotactic responsiveness to its specific ligand SDF1α. Methods Three breast cancer cell lines MDA-MB-231, MCF7 and 4T1 were subjected to 48 hrs of hypoxia or normoxia. Cell surface receptor expression was evaluated using flow cytometry. An extracellular matrix invasion assay and microporous migration assay was used to assess chemotactic response and metastatic ability. Results CXCR4 surface expression was significantly increased in the two human breast cancer cell lines, MDA-MB-231 and MCF7, following exposure to hypoxia. This upregulation of CXCR4 cell surface expression corresponded to a significant increase in migration and invasion in response to SDF1-α in vitro. The increase in metastatic potential of both the normoxic and the hypoxic treated breast cancer cell lines was attenuated by neutralization of CXCR4 with a CXCR4 neutralizing mAb, MAB172 or a CXCR4 antagonist, AMD3100, showing the relationship between CXCR4 overexpression and increased chemotactic responsiveness. Conclusions CXCR4 expression can be modulated by the tissue microenvironment such as hypoxia. Upregulation of CXCR4 is associated with increased migratory and invasive potential and this effect can be abrogated by CXCR4 inhibition. Chemokine receptor CXCR4 is a potential therapeutic target in the adjuvant treatment of breast cancer.

  20. Impact of Pancreatic Rat Islet Density on Cell Survival during Hypoxia

    Directory of Open Access Journals (Sweden)

    A. Rodriguez-Brotons

    2016-01-01

    Full Text Available In bioartificial pancreases (BP, the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2 in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ/cm2 and cultured in normal atmospheric pressure (160 mmHg as well as hypoxic conditions (15 mmHg for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance.

  1. PPARδ deficiency disrupts hypoxia-mediated tumorigenic potential of colon cancer cells.

    Science.gov (United States)

    Jeong, Eunshil; Koo, Jung Eun; Yeon, Sang Hyeon; Kwak, Mi-Kyoung; Hwang, Daniel H; Lee, Joo Young

    2014-11-01

    Peroxisome proliferator-activated receptor (PPAR) δ is highly expressed in colon epithelial cells and closely linked to colon carcinogenesis. However, the role of PPARδ in colon cancer cells in a hypoxic tumor microenvironment is not fully understood. We found that expression of the tumor-promoting cytokines, IL-8 and VEGF, induced by hypoxia (colon cancer cells. Consequently, PPARδ-knockout colon cancer cells exposed to hypoxia and deferoxamine failed to stimulate endothelial cell vascularization and macrophage migration/proliferation, whereas wild-type cells were able to induce angiogenesis and macrophage activation in response to hypoxic stress. Hypoxic stress induced transcriptional activation of PPARδ, but not its protein expression, in HCT116 cells. Exogenous expression of p300 potentiated deferoxamine-induced PPARδ transactivation, while siRNA knockdown of p300 abolished hypoxia- and deferoxamine-induced PPARδ transactivation. PPARδ associated with p300 upon hypoxic stress as demonstrated by coimmunoprecipitation studies. PI3K inhibitors or siRNA knockdown of Akt suppressed the PPARδ transactivation induced by hypoxia and deferoxamine in HCT116 cells, leading to decreased expression of IL-8 and VEGF. Collectively, these results reveal that PPARδ is required for hypoxic stress-mediated cytokine expression in colon cancer cells, resulting in promotion of angiogenesis, macrophage recruitment, and macrophage proliferation in the tumor microenvironment. p300 and the PI3K/Akt pathway play a role in the regulation of PPARδ transactivation induced by hypoxic stress. Our results demonstrate the positive crosstalk between PPARδ in tumor cells and the hypoxic tumor microenvironment and provide potential therapeutic targets for colon cancer. © 2014 Wiley Periodicals, Inc.

  2. Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition.

    Science.gov (United States)

    Yamazaki, Hiroki; Lai, Yu-Chang; Tateno, Morihiro; Setoguchi, Asuka; Goto-Koshino, Yuko; Endo, Yasuyuki; Nakaichi, Munekazu; Tsujimoto, Hajime; Miura, Naoki

    2017-01-01

    We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.

  3. Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes.

    Science.gov (United States)

    Hockman, Dorit; Burns, Alan J; Schlosser, Gerhard; Gates, Keith P; Jevans, Benjamin; Mongera, Alessandro; Fisher, Shannon; Unlu, Gokhan; Knapik, Ela W; Kaufman, Charles K; Mosimann, Christian; Zon, Leonard I; Lancman, Joseph J; Dong, P Duc S; Lickert, Heiko; Tucker, Abigail S; Baker, Clare V H

    2017-04-07

    The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes - carotid body glomus cells, and 'pulmonary neuroendocrine cells' (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive 'neuroepithelial cells' (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.

  4. Hypoxia increases tumor cell shedding of MHC class I chain-related molecule: role of nitric oxide.

    Science.gov (United States)

    Siemens, D Robert; Hu, Nianping; Sheikhi, Abdol Karim; Chung, Eugene; Frederiksen, Lisa J; Pross, Hugh; Graham, Charles H

    2008-06-15

    The MHC class I chain-related (MIC) molecules play important roles in tumor immune surveillance through their interaction with the NKG2D receptor on natural killer and cytotoxic T cells. Thus, shedding of the MIC molecules from the tumor cell membrane represents a potential mechanism of escape from NKG2D-mediated immune surveillance. Tumor hypoxia is associated with a poor clinical outcome for cancer patients. We show that hypoxia contributes to tumor cell shedding of MIC through a mechanism involving impaired nitric oxide (NO) signaling. Whereas hypoxia increased MIC shedding in human prostate cancer cells, activation of NO signaling inhibited hypoxia-mediated MIC shedding. Similar to incubation in hypoxia, pharmacologic inhibition of endogenous NO signaling increased MIC shedding. Parallel studies showed hypoxia-mediated tumor cell resistance to lysis by interleukin 2-activated peripheral blood lymphocytes (PBL) and NO-mediated attenuation of this resistance to lysis. Inhibition of NO production also led to resistance to PBL-mediated lysis. Interference of MIC-NKG2D interaction with a blocking anti-MIC antibody abrogated the effect of hypoxia and NO signaling on tumor cell sensitivity to PBL-mediated lysis. Finally, continuous transdermal delivery of the NO mimetic glyceryl trinitrate (7.3 mug/h) attenuated the growth of xenografted MIC-expressing human prostate tumors. These findings suggest that the hypoxic tumor microenvironment contributes to resistance to immune surveillance and that activation of NO signaling is of potential use in cancer immunotherapy.

  5. 2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study

    Directory of Open Access Journals (Sweden)

    Karina Zitta

    2018-01-01

    Full Text Available Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy (HIE, but additional pharmacological strategies are being explored to further reduce the damage after perinatal asphyxia. The aim of this study was to evaluate whether 2-iminobiotin (2-IB superimposed on hypothermia has the potential to attenuate hypoxia-induced injury of neuronal cells. In vitro hypoxia was induced for 7 h in neuronal IMR-32 cell cultures. Afterwards, all cultures were subjected to 25 h of hypothermia (33.5°C, and incubated with vehicle or 2-IB (10, 30, 50, 100, and 300 ng/ml. Cell morphology was evaluated by brightfield microscopy. Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS was measured using fluorometric assays. Western blotting for PARP, Caspase-3, and the phosphorylated forms of akt and erk1/2 was conducted. To evaluate early apoptotic events and signaling, cell protein was isolated 4 h post-hypoxia and human apoptosis proteome profiler arrays were performed. Twenty-five hour after the hypoxic insult, clear morphological signs of cell damage were visible and significant LDH release as well as ROS production were observed even under hypothermic conditions. Post-hypoxic application of 2-IB (10 and 30 ng/ml reduced the hypoxia-induced LDH release but not ROS production. Phosphorylation of erk1/2 was significantly increased after hypoxia, while phosphorylation of akt, protein expression of Caspase-3 and cleavage of PARP were only slightly increased. Addition of 2-IB did not affect any of the investigated proteins. Apoptosis proteome profiler arrays performed with cellular protein obtained 4 h after hypoxia revealed that post-hypoxic application of 2-IB resulted in a ≥ 25% down regulation of 10/35 apoptosis-related proteins: Bad, Bax, Bcl-2, cleaved Caspase-3, TRAILR1, TRAILR2, PON2, p21, p27, and phospho

  6. An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

    Directory of Open Access Journals (Sweden)

    Ying Huang

    2013-11-01

    Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

  7. Hypoxia/reoxygenation increases the permeability of endothelial cell monolayers: Role of oxygen radicals

    International Nuclear Information System (INIS)

    Inauen, W.; Payne, D.K.; Kvietys, P.R.; Granger, D.N.

    1990-01-01

    We assessed the effect of hypoxia/reoxygenation on 14C-albumin flux across endothelial monolayers. Cultured bovine pulmonary artery endothelial cells were grown to confluence on nitrocellulose filters (pore size 12 microns). The endothelialized filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts (33 nM) of 14C-labeled and unlabeled albumin were added to the hot and cold chambers, respectively. The monolayers were then exposed to successive periods (90 min) of normoxia (pO2 145 mmHg), hypoxia (pO2 20 mmHg), and reoxygenation (pO2 145 mmHg). A gas bubbling system was used to control media pO2 and to ensure adequate mixing. Four aliquots of culture media were taken during each period in order to calculate the 14C-albumin permeability across the endothelialized filter. In some experiments, either the xanthine oxidase inhibitor, oxypurinol (10 microM), or superoxide dismutase (600 U/mL), was added to the media immediately prior to the experiments. As compared to the normoxic control period, albumin permeability was 1.5 times higher during hypoxia (p less than 0.01) and 2.3 times higher during reoxygenation (p less than 0.01). The reoxygenation-induced increase in albumin permeability was prevented by either oxypurinol or superoxide dismutase. These data indicate that xanthine oxidase-derived oxygen radicals contribute to the hypoxia/reoxygenation-induced endothelial cell dysfunction. The altered endothelial barrier function induced by hypoxia/reoxygenation is consistent with the microvascular dysfunction observed following reperfusion of ischemic tissues

  8. The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

    Science.gov (United States)

    Labrecque, Mark P.; Takhar, Mandeep K.; Nason, Rebecca; Santacruz, Stephanie; Tam, Kevin J.; Massah, Shabnam; Haegert, Anne; Bell, Robert H.; Altamirano-Dimas, Manuel; Collins, Colin C.; Lee, Frank J.S.; Prefontaine, Gratien G.; Cox, Michael E.; Beischlag, Timothy V.

    2016-01-01

    Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. PMID:27015368

  9. Hypoxia Responsive, Tumor Penetrating Lipid Nanoparticles for Delivery of Chemotherapeutics to Pancreatic Cancer Cell Spheroids.

    Science.gov (United States)

    Kulkarni, Prajakta; Haldar, Manas K; Katti, Preeya; Dawes, Courtney; You, Seungyong; Choi, Yongki; Mallik, Sanku

    2016-08-17

    Solid tumors are often poorly irrigated due to structurally compromised microcirculation. Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells. However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible to the intravenously injected drug carrier or the drug. Considering the designs of the reported nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-responsive lipid undergoes reduction, destabilizing the lipid membrane, and releasing encapsulated drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions.

  10. Hypercapnia accelerates wound healing in endothelial cell monolayers exposed to hypoxia.

    Science.gov (United States)

    Tsuji, Takao; Aoshiba, Kazutetsu; Itoh, Masayuki; Nakamura, Hiroyuki; Yamaguchi, Kazuhiro

    2013-01-01

    While tissue hypoxia is known to play a critical role in the process of vascular injury and repair, the effect of hypercapnia on this process remains uncertain. We investigated whether hypercapnia might influence endothelial cell wound healing under the influence of hypoxia. Monolayers of human umbilical venous endothelial cells (HUVECs) were scratch-wounded and incubated under different levels of O2, CO2, and pH in the environment. Inhibition of wound healing was observed in the HUVEC monolayers under the hypoxic condition as compared to the normoxic condition. Both hypercapnic acidosis and buffered hypercapnia, but not normocapnic acidosis improved the rate of wound healing under the influence of hypoxia. The beneficial effect of hypercapnia was associated with stimulation of cell proliferation, without effects on cell adhesion, migration or apoptosis. On the other hand, the stimulatory effect of hypercapnia on wound healing and cell proliferation was not noted under normoxic conditions. These results suggest that hypercapnia, rather than acidosis per se, accelerated the wound healing in HUVEC monolayers cultured under hypoxic conditions. The effect of hypercapnia on wound healing was due, at least in part, to the stimulation of cell proliferation by hypercapnia.

  11. [Inhibitory effect of taurine in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation and signal transduction mechanism].

    Science.gov (United States)

    Zhang, Xiao-Dan; Sun, Peng; Zhu, Da-Ling; Xie, Nan

    2014-05-01

    To discuss the effect of taurine (Tau) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs), and study whether the extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway participated in the Tau-inhibited PASMC proliferation process and the possible molecular mechanism. The primary culture was performed for PASMCs in rats. The second to fifth generations were adopted for the experiment. The Tau concentration was 80 mmol x L(-1). The concentration of ERK1/2 blocker (PD98059) was 50 micromol x L(-1). The drug administration time was 24 h. The effect of Tau on the PASMC proliferation was detected by MTT assay, immunofluorescence staining method and western blot under different conditions. The PASMCs were growing were divided into four groups: the normoxia group, the normoxia + Tau group, the hypoxia group and the hypoxia + Tau group. The Western blot was adopted to detect whether the ERK1/2 signal pathway participated in the Tau-inhibited PASMC proliferation process. Subsequently, the PASMCs were divided into five groups: the normoxia group, the hypoxia group, the hypoxia + Tau group, the hypoxia + Tau + PD98059 group and the hypoxia + PD98059 group. Hypoxia could induce the PASMC proliferation. Under the conditions of normoxia, Tau had no effect on the PASMC proliferation. Under the conditions of normoxia and hypoxia, Tau had no effect on the expression of the tumor necrosis factor-alpha (TNF-alpha) among PASMCs. Tau could reverse the expression up-regulation of hypoxia-induced proliferative cell nuclear antigen (PCNA) (P effect on the expression of phosphoryl extracellular signal-regulated kinase 1/2 (p-ERK1/2). Hypoxia could up-regulate the p-ERK1/2 expression (P < 0.01). Tau could reverse the up-regulation of the hypoxia-induced p-ERK1/2 expression(P < 0.01). Both PD98059 and Tau could inhibit the up-regulated expressions of PCNA, Cyclin A and p-ERK1/2. According to the comparison between the single addition of Tau and PD

  12. Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells.

    Science.gov (United States)

    Zeng, Ling; Zhou, Hai-Yun; Tang, Na-Na; Zhang, Wei-Feng; He, Gui-Jun; Hao, Bo; Feng, Ya-Dong; Zhu, Hong

    2016-05-28

    To investigate the influence of phosphatidylinositol-3-kinase protein kinase B (PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, mRNA and activity levels of hypoxia inducible factor-1 alpha (HIF-1α), glucose transporter 1, hexokinase-II, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting siRNA to assess impact of the high expression of HIF-1α on glycolysis. HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymes and the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions. The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.

  13. Aromatic Hydrocarbon Receptor Suppresses Prostate Cancer Bone Metastasis Cells-Induced Vasculogenesis of Endothelial Progenitor Cells under Hypoxia

    Directory of Open Access Journals (Sweden)

    Shuai Huang

    2016-07-01

    Full Text Available Background/Aims: Hypoxia leads to the development of neovascularization in solid tumor by regulating VEGF expression. Aromatic hydrocarbon receptor (AHR, a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with hypoxia-inducible factors 1β (HIF-1β and inhibits the secretion of vascular endothelial growth factor (VEGF. The purpose of this study was to explore whether AHR can suppress hypoxia-induced VEGF production in prostate bone metastasis cells and repress neovascularization in endothelial progenitor cells (EPCs, and, if so, through what mechanisms. Methods: PC-3 or LNCaP cells induced angiogenesis was detected by Matrigel-based tube formation assay, mRNA expression levels was measured by qRT-PCR, VEGF secretion level was determined by ELISA assay, respectively. Results: AHR activation inhibits hypoxia-induced adhesiveness and vasculogenesis of EPCs induced by PC-3 or LNCaP cells under hypoxia. Moreover, AHR activation suppressed hypoxia-induced VEGF production in PC-3 and LNCaP cells (48 ± 14% in PC-3, p = 0.000; 41 ± 14% in LNCaP, p = 0.000 by attenuating HIF-1α and HIF-1β level that in turn diminished the angiogenic ability of EPCs in vitro. Furthermore, we found the mRNA level of hypoxia-inducible factors 1α (HIF-1α (1.54 ± 0.13 fold in PC-3, p = 0.002, 1.62 ± 0.12 fold in LNCaP, p = 0.001 and HIF-1β (1.67 ± 0.23 fold in PC-3, p = 0.007; 1.75 ± 0.26 fold in LNCaP, p=0.008 were upregulated in prostate cancer bone metastasis PC-3 and LNCaP cell lines in response to hypoxia, and revealed that the regulation of VEGF by HIF-1α and HIF-1β was possibly mediated by the activation of phosphatidylinositol 3-kinase pathway. Conclusion: By providing a mechanistic insight into the modulation of neovascularization by AHR ligand, we suggest that AHR ligand has a strong potential of being a new therapeutic agent with applications in the field of bone metastatic prostate cancer.

  14. The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α protein levels in endothelial cells under hypoxia.

    Directory of Open Access Journals (Sweden)

    Stefan K Alig

    Full Text Available The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin. SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132 returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

  15. Hypoxia-inducible factor 1–mediated characteristic features of cancer cells for tumor radioresistance

    International Nuclear Information System (INIS)

    Harada, Hiroshi

    2016-01-01

    Tumor hypoxia has been attracting increasing attention in the fields of radiation biology and oncology since Thomlinson and Gray detected hypoxic cells in malignant solid tumors and showed that they exert a negative impact on the outcome of radiation therapy. This unfavorable influence has, at least partly, been attributed to cancer cells acquiring a radioresistant phenotype through the activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1). On the other hand, accumulating evidence has recently revealed that, even though HIF-1 is recognized as an important regulator of cellular adaptive responses to hypoxia, it may not become active and induce tumor radioresistance under hypoxic conditions only. The mechanisms by which HIF-1 is activated in cancer cells not only under hypoxic conditions, but also under normoxic conditions, through cancer-specific genetic alterations and the resultant imbalance in intermediate metabolites have been summarized herein. The relevance of the HIF-1–mediated characteristic features of cancer cells, such as the production of antioxidants through reprogramming of the glucose metabolic pathway and cell cycle regulation, for tumor radioresistance has also been reviewed

  16. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Soumya C, E-mail: chidambaram.soumya@gmail.com [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Kannan, Anbarasu [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Gopal, Ashidha [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Devaraj, Niranjali [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Halagowder, Devaraj [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India)

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  17. A Novel Chip for Cyclic Stretch and Intermittent Hypoxia Cell Exposures Mimicking Obstructive Sleep Apnea

    Directory of Open Access Journals (Sweden)

    Noelia Campillo

    2016-07-01

    Full Text Available Intermittent hypoxia (IH, a hallmark of obstructive sleep apnea (OSA, plays a critical role in the pathogenesis of OSA-associated morbidities, especially in the cardiovascular and respiratory systems. Oxidative stress and inflammation induced by IH are suggested as main contributors of end-organ dysfunction in OSA patients and animal models. Since the molecular mechanisms underlying these in vivo pathological responses remain poorly understood, implementation of experimental in vitro cell-based systems capable of inducing high-frequency IH would be highly desirable. Here, we describe the design, fabrication and validation of a versatile chip for subjecting cultured cells to fast changes in gas partial pressure and to cyclic stretch. The chip is fabricated with polydimethylsiloxane (PDMS and consists of a cylindrical well covered by a thin membrane. Cells cultured on top of the membrane can be subjected to fast changes in oxygen concentration (equilibrium time 6 s. Moreover, cells can be subjected to cyclic stretch at cardiac or respiratory frequencies independently or simultaneously. Rat bone marrow-derived mesenchymal stem cells (MSCs exposed to IH mimicking OSA and cyclic stretch at cardiac frequencies revealed that hypoxia-inducible factor 1α (HIF-1α expression was increased in response to both stimuli. Thus, the chip provides a versatile tool for the study of cellular responses to cyclical hypoxia and stretch.

  18. A Novel Chip for Cyclic Stretch and Intermittent Hypoxia Cell Exposures Mimicking Obstructive Sleep Apnea.

    Science.gov (United States)

    Campillo, Noelia; Jorba, Ignasi; Schaedel, Laura; Casals, Blai; Gozal, David; Farré, Ramon; Almendros, Isaac; Navajas, Daniel

    2016-01-01

    Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), plays a critical role in the pathogenesis of OSA-associated morbidities, especially in the cardiovascular and respiratory systems. Oxidative stress and inflammation induced by IH are suggested as main contributors of end-organ dysfunction in OSA patients and animal models. Since the molecular mechanisms underlying these in vivo pathological responses remain poorly understood, implementation of experimental in vitro cell-based systems capable of inducing high-frequency IH would be highly desirable. Here, we describe the design, fabrication, and validation of a versatile chip for subjecting cultured cells to fast changes in gas partial pressure and to cyclic stretch. The chip is fabricated with polydimethylsiloxane (PDMS) and consists of a cylindrical well-covered by a thin membrane. Cells cultured on top of the membrane can be subjected to fast changes in oxygen concentration (equilibrium time ~6 s). Moreover, cells can be subjected to cyclic stretch at cardiac or respiratory frequencies independently or simultaneously. Rat bone marrow-derived mesenchymal stem cells (MSCs) exposed to IH mimicking OSA and cyclic stretch at cardiac frequencies revealed that hypoxia-inducible factor 1α (HIF-1α) expression was increased in response to both stimuli. Thus, the chip provides a versatile tool for the study of cellular responses to cyclical hypoxia and stretch.

  19. Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype.

    Science.gov (United States)

    Wang, Fang; Zachar, Vladimir; Pennisi, Cristian Pablo; Fink, Trine; Maeda, Yasuko; Emmersen, Jeppe

    2018-02-08

    Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction ( p Cells differentiated in 5% oxygen conditions showed greater contraction effect ( p cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

  20. Hypoxia inducible factor-1α-dependent epithelial to mesenchymal transition under hypoxic conditions in prostate cancer cells.

    Science.gov (United States)

    Li, Mingchuan; Wang, Yong Xing; Luo, Yong; Zhao, Jiahui; Li, Qing; Zhang, Jiao; Jiang, Yongguang

    2016-07-01

    Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer death. Hypoxia is an environmental stimulus that plays an important role in the development and cancer progression especially for solid tumors. The key regulator under hypoxic conditions is stabilized hypoxia-inducible factor (HIF)-1α. In the present study, immune-fluorescent staining, siRNAs, qRT-PC, immunoblotting, cell migration and invasion assays were carried out to test typical epithelial to mesenchymal transition under hypoxia and the key regulators of this process in PC3, a human prostate cancer cell line. Our data demonstrated that hypoxia induces diverse molecular, phenotypic and functional changes in prostate cancer cells that are consistent with EMT. We also showed that a cell signal factor such as HIF-1α, which might be stabilized under hypoxic environment, is involved in EMT and cancer cell invasive potency. The induced hypoxia could be blocked by HIF-1α gene silencing and reoxygenation of EMT in prostate cancer cells, hypoxia partially reversed accompanied by a process of mesenchymal-epithelial reverting transition (MErT). EMT might be induced by activation of HIF-1α-dependent cell signaling in hypoxic prostate cancer cells.

  1. Hypoxia-Mediated Epigenetic Regulation of Stemness in Brain Tumor Cells.

    Science.gov (United States)

    Prasad, Pankaj; Mittal, Shivani Arora; Chongtham, Jonita; Mohanty, Sujata; Srivastava, Tapasya

    2017-06-01

    Activation of pluripotency regulatory circuit is an important event in solid tumor progression and the hypoxic microenvironment is known to enhance the stemness feature of some cells. The distinct population of cancer stem cells (CSCs)/tumor initiating cells exist in a niche and augment invasion, metastasis, and drug resistance. Previously, studies have reported global hypomethylation and site-specific aberrant methylation in gliomas along with other epigenetic modifications as important contributors to genomic instability during glioma progression. Here, we have demonstrated the role of hypoxia-mediated epigenetic modifications in regulating expression of core pluripotency factors, OCT4 and NANOG, in glioma cells. We observe hypoxia-mediated induction of demethylases, ten-eleven-translocation (TET) 1 and 3, but not TET2 in our cell-line model. Immunoprecipitation studies reveal active demethylation and direct binding of TET1 and 3 at the Oct4 and Nanog regulatory regions. Tet1 and 3 silencing assays further confirmed induction of the pluripotency pathway involving Oct4, Nanog, and Stat3, by these paralogues, although with varying degrees. Knockdown of Tet1 and Tet3 inhibited the formation of neurospheres in hypoxic conditions. We observed independent roles of TET1 and TET3 in differentially regulating pluripotency and differentiation associated genes in hypoxia. Overall, this study demonstrates an active demethylation in hypoxia by TET1 and 3 as a mechanism of Oct4 and Nanog overexpression thus contributing to the formation of CSCs in gliomas. Stem Cells 2017;35:1468-1478. © 2017 AlphaMed Press.

  2. Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism

    International Nuclear Information System (INIS)

    Voss, Melanie J; Möller, Mischa F; Powe, Desmond G; Niggemann, Bernd; Zänker, Kurt S; Entschladen, Frank

    2011-01-01

    Some breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines. Migration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested. Hypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration. Hypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients

  3. Hypoxia-induced expression of bradykinin type-2 receptors in endothelial cells triggers NO production, cell migration, and angiogenesis.

    Science.gov (United States)

    Liesmaa, Inka; Leskinen, Hanna K; Kokkonen, Jorma O; Ruskoaho, Heikki; Kovanen, Petri T; Lindstedt, Ken A

    2009-11-01

    Bradykinin receptors are differentially expressed in the coronary vascular endothelium of rat and human hearts during the pathogenesis of heart failure, but the mechanisms responsible for this regulation have remained vague. Here we show by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, that hypoxia triggers the expression of bradykinin type-2 receptors (BK-2Rs) in cultured human coronary artery endothelial cells (HCAECs), in isolated rat cardiac microvascular endothelial cells (RCMECs), and in rat hearts subjected to ligation of the left anterior descending coronary artery. Mild hypoxia (5% O(2)) induced a fourfold temporal increase in BK-2R mRNA expression in HCAECs, which was also observed at the protein level, whereas severe hypoxia (1% O(2)) slightly inhibited the mRNA expression of BK-2Rs. In addition, HOE-140, a BK-2R antagonist, inhibited mRNA and protein expression of BK-2Rs. The BK-2Rs induced by mild hypoxia were biologically active, that is, capable of inducing intracellular production of nitric oxide (NO) upon activation of HCAECs with bradykinin (BK), a response attenuated by HOE-140. In rat hearts recovering from myocardial infarction, BK-2Rs were upregulated in the endothelium of vessels forming at the border zone between fibrotic scar tissue and healthy myocardium. Furthermore, in an in vitro wound-healing assay, RCMEC migration was increased under mild hypoxic culture conditions in the presence of BK and was attenuated with HOE-140. Our present results show that mild hypoxia triggers a temporal expression of functional BK-2Rs in human and rat endothelial cells and support a role for BK-2Rs in hypoxia-induced angiogenesis.

  4. Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia in vitro

    Directory of Open Access Journals (Sweden)

    Emmrich Frank

    2008-02-01

    Full Text Available Abstract Background One of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood (HUCB cells that were already approved for therapeutic efficacy in vivo. However, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. To address the influence of HUCB cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable SH-SY5Y cells. These cells were incubated under an oxygen-reduced atmosphere (O2 Results Hypoxic cultivation of neurons initially induced a rate of 26% ± 13% of apoptosis. Hypoxia also caused an enhanced expression of Caspase-3 and cleaved poly(ADP-ribose polymerase (PARP. Necrosis was only detected in low amounts. Within the next three days rate of apoptosis in untreated hypoxic cultures cumulated to 85% ± 11% (p ≤ 0.001. Specific cytokine (VEGF patterns also suggest anti-apoptotic strategies of neuronal cells. Remarkably, the administration of MNC showed a noticeable reduction of apoptosis rates to levels of normoxic control cultures (7% ± 3%; p ≤ 0.001. In parallel, clustering of administered MNC next to axons and somata of neuronal cells was observed. Furthermore, MNC caused a pronounced increase of chemokines (CCL5; CCL3 and CXCL10. Conclusion We established an in vitro model of neuronal hypoxia that affords the possibility to investigate both, apoptotic neuronal cell death and neuroprotective therapies. Here we employed the therapeutic model to study neuroprotective properties of HUCB cells. We hypothesize that the neuroprotective effect of MNC was due to anti-apoptotic mechanisms related to direct cell-cell contacts with injured neuronal cells and distinct changes in neuroprotective, inflammatory cytokines as well as to the upregulation of chemokines within the co-cultures.

  5. Aged garlic extract and S-allylcysteine prevent apoptotic cell death in a chemical hypoxia model

    Directory of Open Access Journals (Sweden)

    Marisol Orozco-Ibarra

    Full Text Available BACKGROUND: Aged garlic extract (AGE and its main constituent S-allylcysteine (SAC are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2 has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-Ια and up-regulation of HIF-1a-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells RESULTS: We found that CoCl2 induced the stabilization of HIF-1a and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1a stabilization, activity not previously reported for AGE and SAC CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1a and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions

  6. Hypoxia induces an immunodominant target of tuberculosis specific T cells absent from common BCG vaccines.

    Directory of Open Access Journals (Sweden)

    Hannah Priyadarshini Gideon

    2010-12-01

    Full Text Available M. tuberculosis (MTB species-specific antigenic determinants of the human T cell response are important for immunodiagnosis and vaccination. As hypoxia is a stimulus in chronic tuberculosis infection, we analyzed transcriptional profiles of MTB subject to 168 hours of hypoxia to test the hypothesis that upregulation by hypoxia might result in gene products being recognized as antigens. We identified upregulation of two region of difference (RD 11 (Rv2658C and Rv2659c, and one RD2 (Rv1986 absent from commonly used BCG strains. In MTB infected persons, the IL-2 ELISpot response to Rv1986 peptides was several times greater than the corresponding IFN-γ response to the reference immunodominant ESAT-6 or CFP-10 antigens. The IL-2 response was confined to two epitopic regions containing residues 61-80 and 161-180. The biggest population of IL-2 secreting T cells was single cytokine positive central memory T cells. The IL-2 response to live MTB bacilli lacking Rv1986 was significantly lower than the response to wild type or mutant complemented with Rv1986. In addition, the IL-2 response to Rv1986 was significantly lower in HIV-TB co-infected persons than in HIV uninfected persons, and significantly increased during antiretroviral therapy. These findings demonstrate that Rv1986 is an immunodominant target of memory T cells and is therefore of relevance when considering the partial efficacy of currently used BCG vaccines and provide evidence for a clinical trial comparing BCG strains.

  7. Hypoxia Inducible Factor-Stabilizing Bioactive Glasses for Directing Mesenchymal Stem Cell Behavior

    Science.gov (United States)

    Azevedo, Maria M.; Tsigkou, Olga; Nair, Rekha; Jones, Julian R.; Jell, Gavin

    2015-01-01

    Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, proliferation, and recruitment to sites of injury. Materials capable of affecting the MSC oxygen-sensing pathway, independently of the environmental oxygen pressure, are therefore of immense interest to the tissue engineering (TE) and regenerative medicine community. In this study, we describe the evaluation of the effect of hypoxia inducible factor (HIF)-stabilizing bioactive glasses (BGs) on human MSCs. The dissolution products from these hypoxia-mimicking BGs stabilized HIF-1α in a concentration-dependent manner, altered cell proliferation and metabolism, and upregulated a number of genes involved in the hypoxic response (HIF1A, HIF2A, and VHL), MSC survival (SAG and BCL2), extracellular matrix remodeling (MMP1), and angiogenesis (VEGF and PDGF). These HIF-stabilizing materials can therefore be used to improve MSC survival and enhance regeneration in a number of TE strategies. PMID:25167933

  8. Molecular mechanisms underlying synergistic adhesion of sickle red blood cells by hypoxia and low nitric oxide bioavailability.

    Science.gov (United States)

    Gutsaeva, Diana R; Montero-Huerta, Pedro; Parkerson, James B; Yerigenahally, Shobha D; Ikuta, Tohru; Head, C Alvin

    2014-03-20

    The molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD.

  9. Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro.

    Directory of Open Access Journals (Sweden)

    Tina B McKay

    Full Text Available Keratoconus (KC is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both in vitro and ex vivo. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP expression in human corneal fibroblasts from healthy controls (HCFs and KC patients (HKCs in vitro. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O2 or hypoxic (2% O2 conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP

  10. Hypoxia induced impairment of NK cell cytotoxicity against multiple myeloma can be overcome by IL-2 activation of the NK cells.

    Directory of Open Access Journals (Sweden)

    Subhashis Sarkar

    Full Text Available BACKGROUND: Multiple Myeloma (MM is an incurable plasma cell malignancy residing within the bone marrow (BM. We aim to develop allogeneic Natural Killer (NK cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses. METHODS: NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia. RESULTS: Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2 and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4. It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions. CONCLUSIONS: Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions.

  11. Hypoxia simultaneously alters satellite cell-mediated angiogenesis and hepatocyte growth factor expression.

    Science.gov (United States)

    Flann, K L; Rathbone, C R; Cole, L C; Liu, X; Allen, R E; Rhoads, R P

    2014-05-01

    Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. Hepatocyte growth factor (HGF) plays a pivotal role in myogenesis by activating satellite cells (SC) in regenerating muscle and likely plays a role as a contributor to revascularization. Moreover, repair of a functional blood supply is critical to ameliorate tissue ischemia and restore skeletal muscle function, however effects of hypoxia on satellite cell-mediated angiogenesis remain unclear. The objective of this study was to examine the role of HGF and effect of hypoxia on the capacity of satellite cells to promote angiogenesis. To characterize the role of HGF, a microvascular fragment (MVF) culture model coupled with satellite cell conditioned media (CM) was employed. The activity of HGF was specifically blocked in SC CM reducing sprout length compared to control CM. In contrast, MVF sprout number did not differ between control or HGF-deficient SC CM media. Next, we cultured MVF in the presence of CM from satellite cells exposed to normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1α protein abundance and VEGF mRNA abundance but decreased HGF mRNA abundance compared to normoxic satellite cells. Consistent with reduced HGF gene expression, HGF promoter activity decreased during hypoxia. Taken together, this data indicates that hypoxic modulation of satellite cell-mediated angiogenesis involves a reduction in satellite cell HGF expression. © 2013 Wiley Periodicals, Inc.

  12. Sulforaphane reduces molecular response to hypoxia in ovarian tumor cells independently of their resistance to chemotherapy.

    Science.gov (United States)

    Pastorek, Michal; Simko, Veronika; Takacova, Martina; Barathova, Monika; Bartosova, Maria; Hunakova, Luba; Sedlakova, Olga; Hudecova, Sona; Krizanova, Olga; Dequiedt, Franck; Pastorekova, Silvia; Sedlak, Jan

    2015-07-01

    One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1α protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.

  13. Flooding tolerance and cell wall alterations in maize mesocotyl during hypoxia

    Directory of Open Access Journals (Sweden)

    Vitorino Patrícia Goulart

    2001-01-01

    Full Text Available This research aimed to characterize the tolerance to flooding and alterations in pectic and hemicellulose fractions from mesocotyl of maize tolerant to flooding when submitted to hypoxia. In order to characterize tolerance seeds from maize cultivars Saracura BRS-4154 and BR 107 tolerant and sensitive to low oxygen levels, respectively, were set to germinate. Plantlet survival was evaluated during five days after having been submitted to hypoxia. After fractionation with ammonium oxalate 0.5% (w/v and KOH 2M and 4M, Saracura BRS-4154 cell wall was obtained from mesocotyl segments with different damage intensities caused by oxygen deficiency exposure. The cell wall fractions were analyzed by gel filtration and gas chromatography, and also by Infrared Spectrum with Fourrier Transformation (FTIR. The hypoxia period lasting three days or longer caused cell lysis and in advanced stages plant death. The gelic profile from pectic, hemicellulose 2M and 4M fractions from samples with translucid and constriction zone showed the appearance of low molecular weight compounds, similar to glucose. The main neutral sugars in pectic and hemicellulose fractions were arabinose, xilose and mannose. The FTIR spectrum showed a gradual decrease in pectic substances from mesocotyl with normal to translucid and constriction appearance respectively.

  14. Effects of extracellular modulation through hypoxia on the glucose metabolism of human breast cancer stem cells

    Science.gov (United States)

    Yustisia, I.; Jusman, S. W. A.; Wanandi, S. I.

    2017-08-01

    Cancer stem cells have been reported to maintain stemness under certain extracellular changes. This study aimed to analyze the effect of extracellular O2 level modulation on the glucose metabolism of human CD24-/CD44+ breast cancer stem cells (BCSCs). The primary BCSCs (CD24-/CD44+ cells) were cultured under hypoxia (1% O2) for 0.5, 4, 6, 24 and 48 hours. After each incubation period, HIF1α, GLUT1 and CA9 expressions, as well as glucose metabolism status, including glucose consumption, lactate production, O2 consumption and extracellular pH (pHe) were analyzed using qRT-PCR, colorimetry, fluorometry, and enzymatic reactions, respectively. Hypoxia caused an increase in HIF1α mRNA expressions and protein levels and shifted the metabolic states to anaerobic glycolysis, as demonstrated by increased glucose consumption and lactate production, as well as decreased O2 consumption and pHe. Furthermore, we demonstrated that GLUT1 and CA9 mRNA expressions simultaneously increased, in line with HIF1α expression. In conclusion, modulation of the extracellular environment of human BCSCs through hypoxia shifedt the metabolic state of BCSCs to anaerobic glycolysis, which might be associated with GLUT1 and CA9 expressions regulated by HIFlα transcription factor.

  15. Hypoxia inhibits growth, proliferation, and increases response to chemotherapy in retinoblastoma cells.

    Science.gov (United States)

    Yang, Qian; Tripathy, Arushi; Yu, Wayne; Eberhart, Charles G; Asnaghi, Laura

    2017-09-01

    Retinoblastoma is a malignant tumor of the retina and the most frequent intraocular cancer in children. Low oxygen tension (hypoxia) is a common phenomenon in advanced retinoblastomas, but its biological effect on retinoblastoma growth is not clearly understood. Here we studied how hypoxia altered retinoblastoma gene expression and modulated growth and response to chemotherapy. The hypoxic marker lysyl oxidase (LOX) was expressed in 8 of 12 human retinoblastomas analyzed by immunohistochemistry, suggesting that a hypoxic microenvironment is present in up to two thirds of the cases. WERI Rb1 and Y79 retinoblastoma lines were exposed to 1% or 5% pO 2 , cobalt chloride (CoCl 2 ), or to normoxia (21% pO 2 ) for up to 8 days. Both 1% and 5% pO 2 inhibited growth of both lines by more than 50%. Proliferation was reduced by 25-50% when retinoblastoma cells were exposed to 1% vs 21% pO 2 , as determined by Ki67 assay. Surprisingly, Melphalan, Carboplatin, and Etoposide produced greater reduction in growth and survival of hypoxic cells than normoxic ones. Gene expression profile analysis of both lines, exposed for 48 h to 1%, 5%, or 21% pO 2 , showed that glycolysis and glucose transport were the most up-regulated pathways, whereas oxidative phosphorylation was the most down-regulated pathway in hypoxia as compared to normoxia. These data support a role for hypoxia in suppressing growth, proliferation, and enhancing response of retinoblastoma cells to chemotherapy, possibly by impairing energy production through activation of glycolysis and inhibition of mitochondrial respiration. Targeting glucose metabolism or enhancing delivery of chemotherapeutic agents to hypoxic regions may improve treatment of advanced retinoblastomas. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization.

    Science.gov (United States)

    Tejchman, Anna; Lamerant-Fayel, Nathalie; Jacquinet, Jean-Claude; Bielawska-Pohl, Aleksandra; Mleczko-Sanecka, Katarzyna; Grillon, Catherine; Chouaib, Salem; Ugorski, Maciej; Kieda, Claudine

    2017-05-09

    Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

  17. VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells.

    Science.gov (United States)

    Bindra, Ranjit S; Vasselli, James R; Stearman, Robert; Linehan, W Marston; Klausner, Richard D

    2002-06-01

    Renal cell carcinoma is associated with mutation of the von Hippel-Lindau (VHL) tumor suppressor gene. Cell lines derived from these tumors cannot exit the cell cycle when deprived of growth factors, and the ability to exit the cell cycle can be restored by the reintroduction of wild-type protein VHL (pVHL). Here, we report that cyclin D1 is overexpressed and remains inappropriately high in during contact inhibition in pVHL-deficient cell lines. In addition, hypoxia increased the expression of cyclin D1 specifically in pVHL-negative cell lines into which pVHL expression was restored. Hypoxic-induction of cyclin D1 was not observed in other pVHL-positive cell lines. This suggests a model whereby in some kidney cell types, pVHL may regulate a proliferative response to hypoxia, whereas the loss of pVHL leads to constitutively elevated cyclin D1 and abnormal proliferation under normal growth conditions.

  18. Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status

    Directory of Open Access Journals (Sweden)

    de Carvalho Marcelo

    2011-03-01

    Full Text Available Abstract Background In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions. Results Morphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2 versus hypoxia (5% O2 for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1 were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold involved in extracellular matrix assembly (SMOC2, neural and muscle development (NOG, GPR56, SNTG2, LAMA and epithelial development (DMKN. This group described herein for the first time was assigned by the Gene Ontology program to "plasticity". Conclusion The duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.

  19. [EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Yu, Fengxu; Chen, Yongen; Chen, Feng; Xia, Jiyi; Liu, Hongduan; Fu, Yong; Li, Miaoling; Liao, Bin

    2016-04-01

    To investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. LVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3PLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94% N₂, 1% O₂, and 5% CO₂ for 0, 3, 6, 9, and 12 hours (group B1 and group C1). The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t = 17.525, P = 0.013). The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.

  20. Maternally Expressed Gene 3 (MEG3) Enhances PC12 Cell Hypoxia Injury by Targeting MiR-147.

    Science.gov (United States)

    Han, Lili; Dong, Zhiling; Liu, Ningning; Xie, Fei; Wang, Ning

    2017-01-01

    Cerebral ischemia often leads to breakdown of blood-brain barrier (BBB) and vasogenic edema. It remains to be established whether MEG3 is responsible for the hypoxic damage in neural cells. This study aimed to investigate the role of MEG3 in the hypoxia-induced injuries of PC12 cells. The PC12 cells were seeded and cultured under hypoxia and normoxia culture conditions. The cell viability determined by trypan blue exclusion, apoptosis using propidium iodide (PI) and fluorescein isothiocynate (FITC)-conjugated Annexin V staining, cell-migration using a modified two-chamber migration assay with a pore size of 8 µM and invasion using 24-well Millicell Hanging Cell Culture inserts with 8 µM PET membranes. Cell viability, relative migration and relative invasion decreased significantly in PC12 cells injured due to hypoxia as compared to control cells. An increase in apoptosis was also observed. The expression of MEG3 was up-regulated in hypoxia-injured PC12 cells. MEG3 overexpression enhanced hypoxia injuries, while MEG3 suppression attenuated the injuries. Meanwhile, MEG3 negatively regulated miR-147 expression. In addition, we found that the expression of Sox2 was increased in PC12 cells after hypoxia and miR-147 negatively regulated Sox2 expression through targets its 3'-UTR. Interesting, Sox2 activated NF-κB pathway and Wnt/β-catenin pathway in PC12 cells. Considering the observations in our study, we can conclude that MEG3 aggravated the hypoxial injury in PC12 cells by down-regulating miR-147 gene and miR-147 further negatively regulated Sox2 expression. © 2017 The Author(s). Published by S. Karger AG, Basel.

  1. Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

    Directory of Open Access Journals (Sweden)

    Ranera Beatriz

    2012-08-01

    Full Text Available Abstract Background Mesenchymal stem cells (MSCs derived from bone marrow (BM-MSCs and adipose tissue (AT-MSCs are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2. This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. Results At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. Conclusions Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

  2. Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Xiangjun [Department of Urology, Taihe Hospital, Hubei University of Medicine, Hubei (China); Yao, Qisheng, E-mail: yymcyqs@126.com [Department of Urology, Taihe Hospital, Hubei University of Medicine, Hubei (China); Sun, Xinbo; Gong, Xiaoxin; Yang, Yong; Chen, Congbo [Department of Urology, Taihe Hospital, Hubei University of Medicine, Hubei (China); Shan, Guang [Department of Urology, Renmin Hospital of Wuhan University, Hubei (China)

    2017-03-01

    Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treated with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells injury

  3. Activator protein-1 (AP-1): a bridge between life and death in lung epithelial (A549) cells under hypoxia.

    Science.gov (United States)

    Yadav, Seema; Kalra, Namita; Ganju, Lilly; Singh, Mrinalini

    2017-12-01

    Activator protein-1 (AP-1) transcription factor plays a central role in hypoxia to modulate the expression of genes that decides the fate of the cell. The aim of the present study was to explore the role of AP-1 subunits in lung epithelial (A549) cells under hypoxia. Cell cycle studies by flow cytometry indicated that cell viability was unaffected by the initial hypoxia exposure (0.5% O 2 at 37 °C) for 6 and 12 h. However, both transient cell cycle arrest and cell death was detected at 24 and 48 h. Flow cytometry and spectrofluorometry data confirmed the increase in ROS levels. Elevated ROS and calcium levels activated the stress-related MAPK signaling cascade. ERK and JNK were activated in early hypoxic exposure (within 6 h), whereas p38 were activated in 48 h of hypoxia. These subtypes further stimulated the subunits of AP-1 at different times of hypoxia exposure to orchestrate different genes responsible for cell proliferation (6 and 12 h) and apoptosis (24 and 48 h). Our results clearly depict the role of AP-1 heterodimer, i.e., p-c-jun/c-fos, p-c-jun/fosB, junD/c-fos, and junD/fosB in cell proliferation/survival by regulating the expression of Bcl-2 and cyclins (D1 and B1) at 6 h and 12 h of hypoxia, whereas junB/Fra-1 heterodimer have important role in apoptosis by regulating the expression of p53, Bax, and cyclin-dependent kinase inhibitors (p16, p21, p27) at 24 h and 48 h of hypoxia. Also, the cell survival signaling pathway NO-AKT interrupted at 24 h and 48 h of hypoxia indicating cell death. In conclusion, hypoxia for different time points activated different subunits of AP-1 that combined to form different heterodimers. These dimers regulated the expression of genes responsible for cell proliferation and apoptosis. Since, AP-1 plays a role in the decisive phenomenon of the cell to choose between proliferation and apoptosis; thus, its subunits or dimers could be a good therapeutic target for many diseases.

  4. Hypoxia disrupts the Fanconi anemia pathway and sensitizes cells to chemotherapy through regulation of UBE2T

    International Nuclear Information System (INIS)

    Ramaekers, Chantal H.M.A.; Beucken, Twan van den; Meng, Alice; Kassam, Shaqil; Thoms, John; Bristow, Robert G.; Wouters, Bradly G.

    2011-01-01

    Background and purpose: Hypoxia is a common feature of the microenvironment of solid tumors which has been shown to promote malignancy and poor patient outcome through multiple mechanisms. The association of hypoxia with more aggressive disease may be due in part to recently identified links between hypoxia and genetic instability. For example, hypoxia has been demonstrated to impede DNA repair by down-regulating the homologous recombination protein RAD51. Here we investigated hypoxic regulation of UBE2T, a ubiquitin ligase required in the Fanconi anemia (FA) DNA repair pathway. Materials and methods: We analysed UBE2T expression by microarray, quantitative PCR and western blot analysis in a panel of cancer cell lines as a function of oxygen concentration. The importance of this regulation was assessed by measuring cell survival in response to DNA damaging agents under normoxia or hypoxia. Finally, HIF dependency was determined using knockdown cell lines and RCC4 cells which constitutively express HIF1α. Results: Hypoxia results in rapid and potent reductions in mRNA levels of UBE2T in a panel of cancer cell lines. Reduced UBE2T mRNA expression is HIF independent and was not due to changes in mRNA or protein stability, but rather reflected reduced promoter activity. Exposure of tumor cells to hypoxia greatly increased their sensitivity to treatment with the interstrand crosslinking (ICL) agent mitomycin C. Conclusions: Exposure to hypoxic conditions down-regulates UBE2T expression which correlates with an increased sensitivity to crosslinking agents consistent with a defective Fanconi anemia pathway. This pathway can potentially be exploited to target hypoxic cells in tumors.

  5. IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression.

    Science.gov (United States)

    Hasan, Arif Ul; Kittikulsuth, Wararat; Yamaguchi, Fuminori; Musarrat Ansary, Tuba; Rahman, Asadur; Shibayama, Yuki; Nakano, Daisuke; Hitomi, Hirofumi; Tokuda, Masaaki; Nishiyama, Akira

    2017-09-15

    Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl 2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl 2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, β-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Hypoxia upregulates Bcl-2 expression and suppresses interferon-gamma induced antiangiogenic activity in human tumor derived endothelial cells.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

    BACKGROUND: Hypoxia in solid tumors potentially stimulates angiogenesis by promoting vascular endothelial growth factor (VEGF) production and upregulating VEGF receptor expression. However, it is unknown whether hypoxia can modulate the effect of anti-angiogenic treatment on tumor-derived endothelium. METHODS: Human tumor-derived endothelial cells (HTDEC) were freshly isolated from surgically removed human colorectal tumors by collagenase\\/DNase digestion and Percol gradient sedimentation. Cell proliferation was assessed by measuring BrdU incorporation, and capillary tube formation was measured using Matrigel. Cell apoptosis was assessed by flow cytometry and ELISA, and Bcl-2 expression was detected by Western blot analysis. RESULTS: Under aerobic culture conditions (5% CO2 plus 21% O2) HTDEC expressed less Bcl-2 and were more susceptible to IFN-gamma-induced apoptosis with significant reductions in both cell proliferation and capillary tube formation, when compared with normal human macrovascular and microvascular EC. Following exposure of HTDEC to hypoxia (5% CO2 plus 2% O2), IFN-gamma-induced cell apoptosis, and antiangiogenic activity (i.e. an inhibition in cell proliferation and capillary tube formation) in HTDEC were markedly attenuated. This finding correlated with hypoxia-induced upregulation of Bcl-2 expression in HTDEC. CONCLUSIONS: These results indicate that hypoxia can protect HTDEC against IFN-gamma-mediated cell death and antiangiogenic activity, and suggest that improvement of tumor oxygenation may potentiate the efficacy of anti-cancer therapies specifically targeting the inhibition of tumor angiogenesis.

  7. Sulforaphane inhibits hypoxia-induced HIF-1α and VEGF expression and migration of human colon cancer cells.

    Science.gov (United States)

    Kim, Dong Hwan; Sung, Bokyung; Kang, Yong Jung; Hwang, Seong Yeon; Kim, Min Jeong; Yoon, Jeong-Hyun; Im, Eunok; Kim, Nam Deuk

    2015-12-01

    The effects of sulforaphane (a natural product commonly found in broccoli) was investigated on hypoxia inducible factor-1α (HIF-1α) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1α protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1α expression in both cell lines. Treatment with sulforaphane inhibited hypoxia-induced vascular endothelial growth factor (VEGF) expression in HCT116 cells. Treatment with sulforaphane modulated the effect of hypoxia on HIF-1α stability. However, degradation of HIF-1α by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1α by sulforaphane was not mediated through AKT and extracellular signal-regulated kinase phosphorylation under hypoxic conditions. Finally, hypoxia-induced HCT116 cell migration was inhibited by sulforaphane. These data suggest that sulforaphane may inhibit human colon cancer progression and cancer cell angiogenesis by inhibiting HIF-1α and VEGF expression. Taken together, these results indicate that sulforaphane is a new and potent chemopreventive drug candidate for treating patients with human colon cancer.

  8. Disrupting Hypoxia-Induced Bicarbonate Transport Acidifies Tumor Cells and Suppresses Tumor Growth.

    Science.gov (United States)

    McIntyre, Alan; Hulikova, Alzbeta; Ledaki, Ioanna; Snell, Cameron; Singleton, Dean; Steers, Graham; Seden, Peter; Jones, Dylan; Bridges, Esther; Wigfield, Simon; Li, Ji-Liang; Russell, Angela; Swietach, Pawel; Harris, Adrian L

    2016-07-01

    Tumor hypoxia is associated clinically with therapeutic resistance and poor patient outcomes. One feature of tumor hypoxia is activated expression of carbonic anhydrase IX (CA9), a regulator of pH and tumor growth. In this study, we investigated the hypothesis that impeding the reuptake of bicarbonate produced extracellularly by CA9 could exacerbate the intracellular acidity produced by hypoxic conditions, perhaps compromising cell growth and viability as a result. In 8 of 10 cancer cell lines, we found that hypoxia induced the expression of at least one bicarbonate transporter. The most robust and frequent inductions were of the sodium-driven bicarbonate transporters SLC4A4 and SLC4A9, which rely upon both HIF1α and HIF2α activity for their expression. In cancer cell spheroids, SLC4A4 or SLC4A9 disruption by either genetic or pharmaceutical approaches acidified intracellular pH and reduced cell growth. Furthermore, treatment of spheroids with S0859, a small-molecule inhibitor of sodium-driven bicarbonate transporters, increased apoptosis in the cell lines tested. Finally, RNAi-mediated attenuation of SLC4A9 increased apoptosis in MDA-MB-231 breast cancer spheroids and dramatically reduced growth of MDA-MB-231 breast tumors or U87 gliomas in murine xenografts. Our findings suggest that disrupting pH homeostasis by blocking bicarbonate import might broadly relieve the common resistance of hypoxic tumors to anticancer therapy. Cancer Res; 76(13); 3744-55. ©2016 AACR. ©2016 American Association for Cancer Research.

  9. Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia.

    Directory of Open Access Journals (Sweden)

    Toshiki Hirakawa

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs, the expression of insulin-like growth factor-1 (IGF1 and IGF1 receptor (IGF1R was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9 was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O2 and hypoxia (1% O2. IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC.

  10. Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Shigeo Saito

    2015-06-01

    Full Text Available Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia, cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches. Reactive oxygen species (ROS have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16Ink4a, which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs. During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs. ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development.

  11. Identification of crucial microRNAs and genes in hypoxia-induced human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Geng Y

    2016-07-01

    Full Text Available Ying Geng,1,* Lili Deng,2,* Dongju Su,1 Jinling Xiao,1 Dongjie Ge,3 Yongxia Bao,1 Hui Jing4 1Department of Respiratory, 2Department of Oncology, The Second Affiliated Hospital of Harbin Medical University, 3Department of Respiratory, The First Hospital of Harbin, 4Department of Emergency, The Second Affiliated Hospital of Harbin Medical University Harbin, Heilongjiang, People’s Republic of China *These authors contributed equally to this work Background: Variations of microRNA (miRNA expression profile in hypoxic lung cancer cells have not been studied so far. Therefore, using miRNA microarray technology, this study aimed to study the miRNA expression profile and investigate the potential crucial miRNAs and their target genes in hypoxia-induced human lung adenocarcinoma cells.Materials and methods: Based on miRNA microarray, miRNA expression profiling of hypoxia-induced lung adenocarcinoma A549 cells was obtained. After identification of differentially expressed miRNAs (DE-miRNAs in hypoxic cells, target genes of DE-miRNAs were predicted, and functional enrichment analysis of targets was conducted. Furthermore, the expression levels of DE-miRNAs and their target genes were validated by real-time quantitative polymerase chain reaction. In addition, using miRNA mimics, the effect of overexpressed DE-miRNAs on A549 cell behaviors (cell proliferation, cell cycle, and apoptosis was evaluated.Results: In total, 14 DE-miRNAs (nine upregulated miRNAs and five downregulated miRNAs were identified in hypoxic cells, compared with normoxic cells. Target genes of both upregulated and downregulated miRNAs were enriched in the functions such as chromatin modification, and pathways such as Wnt signaling pathway and transforming growth factor (TGF-β signaling pathway. The expression levels of several miRNAs and their target genes were confirmed, including hsa-miR-301b/FOXF2, hsa-miR-148b-3p/WNT10B, hsa-miR-769-5p/(SMAD2, ARID1A, and hsa-miR-622. Among them

  12. Cancer drug troglitazone stimulates the growth and response of renal cells to hypoxia inducible factors

    International Nuclear Information System (INIS)

    Taub, Mary

    2016-01-01

    Troglitazone has been used to suppress the growth of a number of tumors through apoptosis and autophagy. However, previous in vitro studies have employed very high concentrations of troglitazone (≥10 −5  M) in order to elicit growth inhibitory effects. In this report, when employing lower concentrations of troglitazone in defined medium, troglitazone was observed to stimulate the growth of primary renal proximal tubule (RPT) cells. Rosiglitazone, like troglitazone, is a thiazolidinedione (TZD) that is known to activate Peroxisome Proliferator Activated Receptor Υ (PPARΥ). Notably, rosiglitazone also stimulates RPT cell growth, as does Υ-linolenic acids, another PPARΥ agonist. The PPARΥ antagonist GW9662 inhibited the growth stimulatory effect of troglitazone. In addition, troglitazone stimulated transcription by a PPAR Response Element/Luciferase construct. These results are consistent with the involvement of PPARΥ as a mediator of the growth stimulatory effect of troglitazone. In a number of tumor cells, the expression of hypoxia inducible factor (HIF) is increased, promoting the expression of HIF inducible genes, and vascularization. Troglitazone was observed to stimulate transcription by a HIF/luciferase construct. These observations indicate that troglitazone not only promotes growth, also the survival of RPT cells under conditions of hypoxia. - Highlights: • Troglitazone and rosiglitazone stimulate renal proximal tubule cell growth. • Troglitazone and linolenic acid stimulate growth via PPARϒ. • Linolenic acid stimulates growth in the presence of fatty acid free serum albumin. • Rosiglitazone stimulates transcription by a HRE luciferase construct.

  13. Cancer drug troglitazone stimulates the growth and response of renal cells to hypoxia inducible factors

    Energy Technology Data Exchange (ETDEWEB)

    Taub, Mary, E-mail: biochtau@buffalo.edu

    2016-03-11

    Troglitazone has been used to suppress the growth of a number of tumors through apoptosis and autophagy. However, previous in vitro studies have employed very high concentrations of troglitazone (≥10{sup −5} M) in order to elicit growth inhibitory effects. In this report, when employing lower concentrations of troglitazone in defined medium, troglitazone was observed to stimulate the growth of primary renal proximal tubule (RPT) cells. Rosiglitazone, like troglitazone, is a thiazolidinedione (TZD) that is known to activate Peroxisome Proliferator Activated Receptor Υ (PPARΥ). Notably, rosiglitazone also stimulates RPT cell growth, as does Υ-linolenic acids, another PPARΥ agonist. The PPARΥ antagonist GW9662 inhibited the growth stimulatory effect of troglitazone. In addition, troglitazone stimulated transcription by a PPAR Response Element/Luciferase construct. These results are consistent with the involvement of PPARΥ as a mediator of the growth stimulatory effect of troglitazone. In a number of tumor cells, the expression of hypoxia inducible factor (HIF) is increased, promoting the expression of HIF inducible genes, and vascularization. Troglitazone was observed to stimulate transcription by a HIF/luciferase construct. These observations indicate that troglitazone not only promotes growth, also the survival of RPT cells under conditions of hypoxia. - Highlights: • Troglitazone and rosiglitazone stimulate renal proximal tubule cell growth. • Troglitazone and linolenic acid stimulate growth via PPARϒ. • Linolenic acid stimulates growth in the presence of fatty acid free serum albumin. • Rosiglitazone stimulates transcription by a HRE luciferase construct.

  14. Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.

    Science.gov (United States)

    Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

    2014-03-01

    Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO

  15. Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

    Directory of Open Access Journals (Sweden)

    Kim Chan

    2007-10-01

    Full Text Available Abstract Background Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5. RGC-5 cells were cultured in a closed hypoxic chamber (5% O2 with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38 and nuclear factor-kappa B (NF-κB were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF, a well-known protective neurotrophin for retinal ganglion cells. Results After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-κB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-κB, while BDNF suppressed phosphorylation of ERK and p38. Conclusion Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-κB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia.

  16. Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma

    Energy Technology Data Exchange (ETDEWEB)

    Tse, Anna Chung-Kwan [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China); Li, Jing-Woei; Chan, Ting-Fung [School of Life Sciences, Hong Kong Bioinformatics Centre, The Chinese University of Hong Kong, Hong Kong SAR (China); Wu, Rudolf Shiu-Sun [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China); Lai, Keng-Po, E-mail: balllai@hku.hk [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China)

    2015-08-15

    Highlights: • We demonstrate hypoxia induced miR-210 in ovarian follicular cells. • We show anti-apoptotic roles of miR-210 in ovarian follicular cells under hypoxia. • Apoptotic genes (DLC1, SLK, TNFRSF10B, RBM25, and USP7) are target of miR-210. • MiR-210 is vital for ovarian follicular cells proliferation in response to hypoxia. - Abstract: Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker – caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of mi

  17. Multifunctional Phosphorescent Conjugated Polymer Dots for Hypoxia Imaging and Photodynamic Therapy of Cancer Cells

    Science.gov (United States)

    Zhou, Xiaobo; Liang, Hua; Jiang, Pengfei; Zhang, Kenneth Yin; Liu, Shujuan; Yang, Tianshe; Yang, Lijuan; Lv, Wen; Yu, Qi

    2015-01-01

    Molecular oxygen (O2) plays a key role in many physiological processes, and becomes a toxicant to kill cells when excited to 1O2. Intracellular O2 levels, or the degree of hypoxia, are always viewed as an indicator of cancers. Due to the highly efficient cancer therapy ability and low side effect, photodynamic therapy (PDT) becomes one of the most promising treatments for cancers. Herein, an early‐stage diagnosis and therapy system is reported based on the phosphorescent conjugated polymer dots (Pdots) containing Pt(II) porphyrin as an oxygen‐responsive phosphorescent group and 1O2 photosensitizer. Intracellular hypoxia detection has been investigated. Results show that cells treated with Pdots display longer lifetimes under hypoxic conditions, and time‐resolved luminescence images exhibit a higher signal‐to‐noise ratio after gating off the short‐lived background fluorescence. Quantification of O2 is realized by the ratiometric emission intensity of phosphorescence/fluorescence and the lifetime of phosphorescence. Additionally, the PDT efficiency of Pdots is estimated by flow cytometry, MTT cell viability assay, and in situ imaging of PDT induced cell death. Interestingly, Pdots exhibit a high PDT efficiency and would be promising in clinical applications. PMID:27722081

  18. Matrix Metalloproteinase 9 Secreted by Hypoxia Cardiac Fibroblasts Triggers Cardiac Stem Cell Migration In Vitro

    Directory of Open Access Journals (Sweden)

    Qing Gao

    2015-01-01

    Full Text Available Cessation of blood supply due to myocardial infarction (MI leads to complicated pathological alteration in the affected regions. Cardiac stem cells (CSCs migration plays a major role in promoting recovery of cardiac function and protecting cardiomyocytes in post-MI remodeling. Despite being the most abundant cell type in the mammalian heart, cardiac fibroblasts (CFs were underestimated in the mechanism of CSCs migration. Our objective in this study is therefore to investigate the migration related factors secreted by hypoxia CFs in vitro and the degree that they contribute to CSCs migration. We found that supernatant from hypoxia induced CFs could accelerate CSCs migration. Four migration-related cytokines were reported upregulated both in mRNA and protein levels. Upon adding antagonists of these cytokines, the number of migration cells significantly declined. When the cocktail antagonists of all above four cytokines were added, the migration cells number reduced to the minimum level. Besides, MMP-9 had an important effect on triggering CSCs migration. As shown in our results, MMP-9 induced CSCs migration and the underlying mechanism might involve TNF-α signaling which induced VEGF and MMP-9 expression.

  19. Effects of hypoxia on expression of a panel of stem cell and chemoresistance markers in glioblastoma-derived spheroids

    DEFF Research Database (Denmark)

    Kolenda, Jesper; Jensen, Stine Skov; Aaberg-Jessen, Charlotte

    2011-01-01

    ). Spheroids were formed in 21% and 1% O(2) in serum-free medium. The immunohistochemical panel included hypoxia (HIF-1α, HIF-2α), proliferation (Ki-67), and stem cell markers (CD133, podoplanin, Bmi-1, nestin, Sox-2) as well as markers related to chemoresistance (MGMT, TIMP-1, Lamp-1, MRP1, MDR-1....... Expression of stem cell markers CD133, podoplanin, Bmi-1, and nestin was increased in hypoxia, whereas Sox-2 was increased in SJ-1 only. TIMP-1 and Lamp-1 were increased in both SJ-1 and U87. In conclusion, the tumor cell phenotype related to stemness, and thereby potentially to chemoresistance, seems...

  20. Hypofractionation results in reduced tumor cell kill compared to conventional fractionation for tumors with regions of hypoxia.

    Science.gov (United States)

    Carlson, David J; Keall, Paul J; Loo, Billy W; Chen, Zhe J; Brown, J Martin

    2011-03-15

    Tumor hypoxia has been observed in many human cancers and is associated with treatment failure in radiation therapy. The purpose of this study is to quantify the effect of different radiation fractionation schemes on tumor cell killing, assuming a realistic distribution of tumor oxygenation. A probability density function for the partial pressure of oxygen in a tumor cell population is quantified as a function of radial distance from the capillary wall. Corresponding hypoxia reduction factors for cell killing are determined. The surviving fraction of a tumor consisting of maximally resistant cells, cells at intermediate levels of hypoxia, and normoxic cells is calculated as a function of dose per fraction for an equivalent tumor biological effective dose under normoxic conditions. Increasing hypoxia as a function of distance from blood vessels results in a decrease in tumor cell killing for a typical radiotherapy fractionation scheme by a factor of 10(5) over a distance of 130 μm. For head-and-neck cancer and prostate cancer, the fraction of tumor clonogens killed over a full treatment course decreases by up to a factor of ∼10(3) as the dose per fraction is increased from 2 to 24 Gy and from 2 to 18 Gy, respectively. Hypofractionation of a radiotherapy regimen can result in a significant decrease in tumor cell killing compared to standard fractionation as a result of tumor hypoxia. There is a potential for large errors when calculating alternate fractionations using formalisms that do not account for tumor hypoxia. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells.

    Science.gov (United States)

    Pradhan, Shrikant; Mahajan, Divyank; Kaur, Prabhjot; Pandey, Namita; Sharma, Chandresh; Srivastava, Tapasya

    2016-11-01

    Non-small cell lung cancer (NSCLC), comprising 85% of lung cancer cases, has been associated with resistance to chemo/radiotherapy. The hypoxic tumor micro-environment, where insufficient vasculature results in poor drug penetrance and sub-optimal chemotherapy in the tumor interiors contributes heavily to this resistance. Additionally, epigenetic changes in tumorigenic cells also change their response to different forms of therapy. In our study, we have investigated the effectiveness of a combination of cisplatin with scriptaid [a pan-Histone Deacetylase inhibitor (HDACi)] in a model that mimics the tumor microenvironment of hypoxia and sub-lethal chemotherapy. Scriptaid synergistically increases the efficacy of cisplatin in normoxia as well as hypoxia, accompanied with reduced metastasis and enhanced DNA damage. Addition of scriptaid also overcomes the cisplatin resistance exhibited in lung cancer cells with stabilized hypoxia inducible factor 1 (HIF1)-α (mutant) and mutant p53. Molecular studies showed that the combination treatment increased apoptotic cell death in both normoxia and hypoxia with a dual role of p38MAPK. Together, our results suggest that the combination of low dose cisplatin and scriptaid is cytotoxic to NSCLC lines, can overcome hypoxia induced resistance and mutant p53- induced instability often associated with this cancer, and has the potential to be an effective therapeutic modality.

  2. Cell and Signal Components of the Microenvironment of Bone Metastasis Are Affected by Hypoxia

    Directory of Open Access Journals (Sweden)

    Paola Bendinelli

    2016-05-01

    Full Text Available Bone metastatic cells release bone microenvironment proteins, such as the matricellular protein SPARC (secreted protein acidic and rich in cysteine, and share a cell signaling typical of the bone metabolism controlled by Runx2. The megakaryocytes in the bone marrow engrafted by the metastases seem to be one of the principal microenvironment sources of the biological stimuli, implicated in the formation of an osteoblastic niche, and affecting metastasis phenotype and colonization. Educated platelets in the circulation might derive from megakaryocytes in bone metastasis. The evaluation of predictive markers in the circulating platelets might be useful for the stratification of patients for therapeutic purposes. The hypoxic environment in bone metastasis is one of the key regulators of the network of the biological soluble and structural components of the matrix. In bone metastatic cells under hypoxia, similar patterns of Runx2 and SPARC are observed, both showing downregulation. Conversely, hypoxia induces Endothelin 1, which upregulates SPARC, and these biological stimuli may be considered prognostic markers of bone metastasis in breast carcinoma patients.

  3. Na(+)/H(+) exchange subtype 1 inhibition during extracellular acidification and hypoxia in glioma cells.

    Science.gov (United States)

    Glunde, Kristine; Düssmann, Heiko; Juretschke, Hans-Paul; Leibfritz, Dieter

    2002-01-01

    Lactacidosis is a common feature of ischaemic brain tissue, but its role in ischaemic neuropathology is still not fully understood. Na(+)/H(+) exchange, a mechanism involved in the regulation of intracellular pH (pH(i)), is activated by low pH(i). The role of Na(+)/H(+) exchange subtype 1 was investigated during extracellular acidification and subsequent pH recovery in the absence and presence of (4-isopropyl-3-methylsulphonyl-benzoyl)-guanidine methanesulfonate (HOE642, Cariporid), a new selective and powerful inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE-1). It was compared for normoxia and hypoxia in two glioma cell lines (C6 and F98). pH(i) was monitored by fluorescence spectroscopy using the intracellularly trapped pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Alterations in glial cell metabolism were characterized using high-resolution (1)H, (13)C and (31)P NMR spectroscopy of perchloric acid extracts. NHE-1 contributed to glial pH regulation, especially at pathologically low pH(i) values. NHE-1 inhibition with HOE642 during acidification caused exacerbated metabolic disorders which were prolonged during extracellular pH recovery. However, NHE-1 inhibition during hypoxia protected the energy state of glial cells.

  4. Radiosensitive effect of hypoxia-inducible factor 1α inhibitor YC-1 on hypoxic glioma SHG44 cell line

    International Nuclear Information System (INIS)

    Guo Xinwei; Lu Xueguan; Tong Liumei; Zong Tianzhou; Chen Liesong

    2011-01-01

    Objective: To investigate the radiosensitive effect of hypoxia-inducible factor 1α (HIF-1α) inhibitor YC-1 on hypoxic glioma SHG44 cell line and its related mechanism. Methods: Glioma SHG44 cell line was cultured in normoxic (20% O 2 ), continuous hypoxia (1% O 2 ) for 12 h and 24 h, continuous hypoxia plus YC-1 was performed for 12 h and 24 h, respectively. The expression of HIF-1α was assessed by Western blot. The radiosensitivity was evaluated by the survival curve, and the sublethal damage repair (SLDR) ability was measured by dose-fraction experiment. Results: HIF-1α protein levels of glioma SHG44 cells were significantly increased after hypoxic cultures for 12 h and 24 h than those of the corresponding cells cultured in normoxic, while the radiosensitivity was lower. The OER (oxygen-enhancement ratio) of SHG44 cells in hypoxia for 12 h and 24 h were 1.22 and 1.37, respectively. By the further statistical analysis it was found that SLDR ability of glioma SHG44 was increased at hypoxia, and when irradiation was carried one at the interval of 8, 10, 12 h it was statistically significant (P<0.05). HIF-1α protein levels of glioma SHG44 cells cultured in hypoxia plus YC-1 for 12 h and 24 h were decreased significantly compared to the corresponding cells cultured in hypoxia only, while the radiosensitivity was significantly increased. the EF (enhancement factor) of YC-1 for glioma SHG44 cells at hypoxia for 12 h and 24 h was 1.27. By the further statistical analysis it was also found that SLDR ability was decreased significantly for hypoxic SHG44 cells which was co-cultured with YC-1, and at the interval of 8, 10, 12 h irradiation was statistically significant (P<0.05). Conclusion: YC-1 can increase the radiosensitivity of hypoxic glioma SHG44 cell line, and its mechanism is related to SLDR inhibited by YC-1. (authors)

  5. Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells.

    Science.gov (United States)

    Gawlitta, Debby; van Rijen, Mattie H P; Schrijver, Edmée J M; Alblas, Jacqueline; Dhert, Wouter J A

    2012-10-01

    Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well

  6. The integrin-linked kinase gene up-regulated by hypoxia plays its pro-survival role in colorectal cancer cells.

    Science.gov (United States)

    Xiao, Lei; Yue, Xiaolong; Ming, Xiaodong; Xu, Lishan; Ding, Mingfeng; Xu, Jun; Liu, Qian

    2014-02-01

    Colorectal cancer (CRC) is a leading cause of cancer death in recent years. It is believed that there are hypoxic regions in both early and advanced stage of tumor and hypoxia is able to reinforce the aggressiveness of tumor cells and accelerate the progression of cancer. Until now the mechanisms by which hypoxia promotes the progression of CRC are far from well understood. Integrin-linked kinase (ILK) is a crucial mediator and over-expressed in CRC patients. But whether ILK is involved in the process that hypoxia promotes CRC cells growth and silencing the ILK gene results in CRC cells apoptosis is not clear. Lentivirus transfection, invasion assay, TUNEL assay, Bromodeoxyuridine incorporation and mitochondrial function assay were applied to demonstrate our hypothesis. In this study, we found that hypoxia induced the expression of ILK in a time-dependent manner, and after knocking down ILK expression with ILK shRNA, the cells proliferation promoted by hypoxia was inhibited in HT29 cell line. Moreover, blocking the ILK pathway led to caspase-3 and caspase-9 activations, the decrease of mitochondrial membrane potential, and cells apoptosis. And the inhibitory effects of hypoxia on cells apoptosis were mediated by the ILK pathway. In addition, hypoxia promoted HT29 cells metastasis and invasion through the ILK pathway. Therefore, we conclude that the CRC cells survival and invasion enhanced by hypoxia are mediated by ILK, and ILK may be an important potential therapeutic target for CRC.

  7. Inhibition of Hypoxia-Induced Cell Motility by p16 in MDA-MB-231 Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Liyuan Li, Yi Lu

    2010-01-01

    Full Text Available Our previous studies indicated that p16 suppresses breast cancer angiogenesis and metastasis, and downregulates VEGF gene expression by neutralizing the transactivation of the VEGF transcriptional factor HIF-1α. Hypoxia stimulates tumor malignant progression and induces HIF-1α. Because p16 neutralizes effect of HIF-1α and attenuates tumor metastatic progression, we intended to investigate whether p16 directly affects one or more aspects of the malignant process such as adhesion and migration of breast cancer cells. To approach this aim, MDA-MB-231 and other breast cancer cells stably transfected with Tet-on inducible p16 were used to study the p16 effect on growth, adhesion and migration of the cancer cells. We found that p16 inhibits breast cancer cell proliferation and migration, but has no apparent effect on cell adhesion. Importantly, p16 inhibits hypoxia-induced cell migration in breast cancer in parallel with its inhibition of HIF-1α transactivation activity. This study suggests that p16's ability to suppress tumor metastasis may be partially resulted from p16's inhibition on cell migration, in addition to its known functions on inhibition of cell proliferation, angiogenesis and induction of apoptosis.

  8. Radiation induced bystander effect on hepatoma HepG2 cells under hypoxia condition

    International Nuclear Information System (INIS)

    Zhang Jianghong; Jin Yizun; Shao Chunlin; Prise KM

    2009-01-01

    Objective: To investigate radiation induced bystander effect and its mechanism on hepatoma HepG2 cells under hypoxia condition. Methods: Non-irradiated bystander hepatoma cells were co-cultured with irradiated cells or treated with the conditioned medium (CM) from irradiated cells, then micronuclei (MN) were measured for both irradiated cells and bystander cells. Results: The MN yield of irradiated HepG2 cells under hypoxic condition was significantly lower than that under normoxia, the oxygen enhancement ratio of HepG2 cells of MN was 1.6. For both hypoxic and normoxic condition, the MN yield of bystander cells were obviously enhanced to a similar high level after co-culturing with irradiated cells or with CM treatment, and it also correlated with the irradiation dose. When the hypoxic HepG2 cells were treated with either DMSO, a scavenger of reactive oxygen species (ROS), or aminoguanidine, an iNOS inhibitor, the yield of bystander MN was partly diminished, and the reducing rate of DMSO was 42.2%-46.7%, the reducing rate of aminoguanidine was 42% . Conclusion: ROS, NO and their downstream signal factors are involved in the radiation induced bystander effect of hypoxic HepG2 cells. (authors)

  9. Effects of Hypoxia on the Immunomodulatory properties of Adipose tissue-derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    M eRoemeling-Van Rhijn

    2013-07-01

    Full Text Available Adipose tissue-derived mesenchymal stem cells (ASC are of great interest as a cellular therapeutic agent for regenerative and immunomodulatory purposes. The function of ASC adapts to environmental conditions, such as oxygen tension. Oxygen levels within tissues are typically much lower than under standard culture conditions and ASC used for therapy therefore encounter a change from normoxic to hypoxic conditions. The effect of hypoxia on the regenerative potential of ASC has been investigated in a number of studies. The effect of hypoxia on the immunomodulatory function of ASC, however, remains to be determined. In the present study the effect of hypoxic (1% oxygen culture conditions on human ASC was examined. ASC showed no signs of toxicity under low oxygen levels and no major immunophenotypical changes were observed, apart from a down regulation of the marker CD105. Oxygen tension had no effect on the proliferation of ASC and colony forming unit (CFU efficiency remained the same under 1% and 20% oxygen. Under both oxygen levels ASC were capable of strong upregulation of the immunomodulatory molecules indolamine 2,3-dioxygenase (IDO and programmed death ligand-1 (PD-L1 upon stimulation with IFN- and TNF-, and, in addition, IDO activity as measured by the accumulation of L-kynurenine was not affected under hypoxia. The ability of ASC to inhibit anti-CD3/CD28 stimulated CD4+ and CD8+ T cell proliferation was enhanced under hypoxic conditions.The results of the present study demonstrate that the immunosuppressive capacity of ASC is maintained under hypoxic conditions. These findings are important for the therapeutic use of ASC and may be applied for the in vitro generation of ASC with improved functionality for therapeutic use.

  10. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

    Science.gov (United States)

    Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

    2016-07-30

    This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

  11. Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia.

    Science.gov (United States)

    Ophelders, Daan R M G; Wolfs, Tim G A M; Jellema, Reint K; Zwanenburg, Alex; Andriessen, Peter; Delhaas, Tammo; Ludwig, Anna-Kristin; Radtke, Stefan; Peters, Vera; Janssen, Leon; Giebel, Bernd; Kramer, Boris W

    2016-06-01

    Preterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells. Bone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration

  12. MicroRNA-145 Aggravates Hypoxia-Induced Injury by Targeting Rac1 in H9c2 Cells.

    Science.gov (United States)

    Wang, Ximing; Zhang, Yanxia; Wang, Hongshan; Zhao, Genshang; Fa, Xianen

    2017-01-01

    Myocardial infarction (MI) is a leading cause of morbidity and mortality. Here, we sought to explore the potential role and underlying mechanism of miR-145 in MI. H9c2 cells were cultured under persistent hypoxia to simulate MI. The hypoxia-induced injury was assessed on the basis of cell viability, migration, invasion and apoptosis. The expression of miR-145 was evaluated by qRT-PCR and the influence of aberrantly expressed miR-145 on H9c2 cells under hypoxia was also estimated. Utilizing bioinformatics methods, the target genes of miR-145 were verified by luciferase reporter assay. Then, effects of abnormally expressed target gene on miR-145 silenced H9c2 cells were assessed. Finally, the phosphorylation levels of key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways were detected by Western blot analysis. Hypoxia remarkably lowered viability, migration and invasion but promoted cell apoptosis. Meantime, the miR-145 level was up-regulated in H9c2 cells under hypoxia. Following experiments suggested that hypoxia-induced injury was exacerbated by miR-145 overexpression while was alleviated by miR-145 silence. Rac1 was predicted and further validated to be a target gene of miR-145. The influence of miR-145 silencing on H9c2 cells under hypoxia could be reversed by down-regulation of Rac1. Additionally, the phosphorylation levels of PI3K, AKT, MAPK and ERK were all elevated in miR-145 silenced cells and these alterations were reversed by down-regulation of Rac1. miR-145 silencing could protect H9c2 cells against hypoxia-induced injury by targeting Rac1, in which PI3K/AKT and MAPK/ERK pathways might be involved. © 2017 The Author(s). Published by S. Karger AG, Basel.

  13. An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption.

    Science.gov (United States)

    Minoves, Mélanie; Morand, Jessica; Perriot, Frédéric; Chatard, Morgane; Gonthier, Brigitte; Lemarié, Emeline; Menut, Jean-Baptiste; Polak, Jan; Pépin, Jean-Louis; Godin-Ribuot, Diane; Briançon-Marjollet, Anne

    2017-10-01

    Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O 2 followed by 1 min at 2% O 2 resulted in Po 2 values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po 2 variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po 2 variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA. Copyright © 2017 the American Physiological Society.

  14. Hypoxia regulates the expression and localization of CCAAT/enhancer binding protein α by hypoxia inducible factor-1α in bladder transitional carcinoma cells.

    Science.gov (United States)

    Xue, Mei; Li, Xu; Chen, Wei

    2015-08-01

    Hypoxia inducible factor-1α (HIF-1α) is overexpressed in various types of solid tumor in humans, including bladder cancer. HIF-1α regulates the expression of a series of genes, which are involved in cell proliferation, differentiation, apoptosis, angiogenesis, migration and invasion and represents a potential therapeutic target for the treatment of human cancer. Despite extensive investigation of the effects of HIF-1α in the progression and metastasis of bladder cancer, the possible regulatory mechanisms underlying the effects of HIF-1α on bladder cancer cell proliferation and differentiation remain to be elucidated. It has been suggested that the transcription factor CCAAT/enhancer binding protein α (C/EBPα) acts as a tumor suppressor in several types of cancer cell, which are involved in regulating cell differentiation, proliferation and apoptosis. The present study confirmed that, in bladder cancer cells, the expression and localization of C/EBPα was regulated by hypoxia through an HIF-1α -dependent mechanism, which may be significant in bladder cancer cell proliferation and differentiation. The 5637 and T24 bladder cancer cell lines were incubated under normoxic and hypoxic conditions. The expression levels of HIF-1α and C/EBPα were detected by reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence analysis. The results revealed that, under hypoxic conditions, the protein expression levels of HIF-1α were markedly upregulated, but the mRNA levels were not altered. However, the mRNA and protein levels of C/EBPα were significantly reduced. The present study further analyzed the subcellular localization of C/EBPα, which was markedly decreased in the nuclei under hypoxic conditions. Following HIF-1α small interference RNA silencing of HIF-1α, downregulation of C/EBPα was prevented in the bladder cancer cells cultured under hypoxic conditions. In addition, groups of cells treated with 3-(5'-hydroxymethyl

  15. The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

    Science.gov (United States)

    Gerber, Philipp A; Rutter, Guy A

    2017-04-01

    Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

  16. Transcription Factor Brn-3b Overexpression Enhances Neurite Outgrowth in PC12 Cells Under Condition of Hypoxia.

    Science.gov (United States)

    Phatak, Nitasha R; Stankowska, Dorota L; Krishnamoorthy, Raghu R

    2015-08-01

    Transcription factor Brn-3b plays a key role in retinal ganglion cell differentiation, survival, and axon outgrowth during development. However, the precise role of Brn-3b in the normal adult retina as well as during neurodegeneration is unclear. In the current study, the effect of overexpression of Brn-3b was assessed in vitro, in PC12 cells under conditions of normoxia and hypoxia. Immunoblot analysis showed that overexpression of Brn-3b in PC12 cells as well as 661W cells produced significant increase in the growth cone marker, growth-associated protein-43 (GAP-43), and acetylated-tubulin (ac-TUBA). In addition, an increased immunostaining for GAP-43 and ac-TUBA was observed in PC12 cells overexpressing Brn-3b, which was accompanied by a marked increase in neurite outgrowth, compared to PC12 cells overexpressing the empty vector. In separate experiments, one set of PC12 cells transfected either with a Brn-3b expression vector or an empty vector was subjected to conditions of hypoxia for 2 h, while another set of similarly transfected PC12 cells was maintained in normoxic conditions. It was found that the upregulation of GAP-43 and ac-TUBA in PC12 cells overexpressing Brn-3b under conditions of normoxia was sustained under conditions of hypoxia. Immunocytochemical analysis revealed not only an upregulation of GAP-43 and ac-TUBA, but also increased neurite outgrowth in PC12 cells transfected with Brn-3b as compared to PC12 cells transfected with empty vector in both normoxia and hypoxia. The findings have implications for a potential role of Brn-3b in neurodegenerative diseases in which hypoxia/ischemia contribute to pathophysiology of the disease.

  17. Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Tanya L Medley

    Full Text Available BACKGROUND: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes. OBJECTIVE: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha and the canonical Wnt pathway in this process. METHODS: Embryoid bodies (EBs derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture. RESULTS: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway. CONCLUSION: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.

  18. Hypoxia. 3. Hypoxia and neurotransmitter synthesis.

    Science.gov (United States)

    Kumar, Ganesh K

    2011-04-01

    Central and peripheral neurons as well as neuroendocrine cells express a variety of neurotransmitters/modulators that play critical roles in regulation of physiological systems. The synthesis of several neurotransmitters/modulators is regulated by O(2)-requiring rate-limiting enzymes. Consequently, hypoxia resulting from perturbations in O(2) homeostasis can affect neuronal functions by altering neurotransmitter synthesis. Two broad categories of hypoxia are frequently encountered: continuous hypoxia (CH) and intermittent hypoxia (IH). CH is often seen during high altitude sojourns, whereas IH is experienced in sleep-disordered breathing with recurrent apneas (i.e., brief, repetitive cessations of breathing). This article presents what is currently known on the effects of both forms of hypoxia on neurotransmitter levels and neurotransmitter synthesizing enzymes in the central and peripheral nervous systems.

  19. The Hypoxia-Mimetic Agent Cobalt Chloride Differently Affects Human Mesenchymal Stem Cells in Their Chondrogenic Potential

    Directory of Open Access Journals (Sweden)

    Gabriella Teti

    2018-01-01

    Full Text Available Adult stem cells are a promising cell source for cartilage regeneration. They resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Cobalt chloride (CoCl2 imitates hypoxia in vitro by stabilizing hypoxia-inducible factor-alpha (HIF-1α, which is the master regulator in the cellular adaptive response to hypoxia. In this study, the influence of CoCl2 on the chondrogenic potential of human MSCs, isolated from dental pulp, umbilical cord, and adipose tissue, was investigated. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 μM. Cell viability, HIF-1α protein synthesis, and the expression of the chondrogenic markers were analyzed. The results showed that the CoCl2 supplementation had no effect on cell viability, while the upregulation of chondrogenic markers such as SOX9, COL2A1, VCAN, and ACAN was dependent on the cellular source. This study shows that hypoxia, induced by CoCl2 treatment, can differently influence the behavior of MSCs, isolated from different sources, in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies.

  20. The stabilization of hypoxia inducible factor modulates differentiation status and inhibits the proliferation of mouse embryonic stem cells.

    Science.gov (United States)

    Binó, Lucia; Kučera, Jan; Štefková, Kateřina; Švihálková Šindlerová, Lenka; Lánová, Martina; Kudová, Jana; Kubala, Lukáš; Pacherník, Jiří

    2016-01-25

    Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

    Directory of Open Access Journals (Sweden)

    Hyou-Ju Jin

    Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

  2. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

    Science.gov (United States)

    Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

  3. Hypoxia-inducible factor-2α-dependent hypoxic induction of Wnt10b expression in adipogenic cells.

    Science.gov (United States)

    Park, Young-Kwon; Park, Bongju; Lee, Seongyeol; Choi, Kang; Moon, Yunwon; Park, Hyunsung

    2013-09-06

    Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O2 concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.

  4. Identification of Appropriate Housekeeping Genes for Gene Expression Analysis in Long-term Hypoxia-treated Kidney Cells.

    Science.gov (United States)

    Moein, Shiva; Javanmard, Shaghayegh Haghjooy; Abedi, Maryam; Izadpanahi, Mohammad Hosein; Gheisari, Yousof

    2017-01-01

    Selection of stably expressing housekeeping genes (HKGs) is a crucial step in gene expression analysis. However, there are no universal HKGs for all experiments, and they should be determined by each biologic condition. The aim of this study was to detect appropriate HKGs for kidney cells cultured in long-term hypoxia. Based on a screening step using a microarray data available from gene expression omnibus database, a set of candidate HKGs were chosen to be assessed in human kidney cells cultured in hypoxic or normoxic conditions for about 2 weeks in a time course manner. The stability of gene expression was assessed by refFinder, a web-based tool that integrates four computational programs (geNorm, Normfinder, BestKeeper, and the comparative ΔΔCt method). GAPDH and ACTB were the most stable genes in hypoxia treated cells whereas, B2M and ACTB were the best HKGs in cells cultured in normoxia. When both hypoxia and normoxia treated cells from all time points were evaluated together, GAPDH and ACTB equally showed the most stability. As in relative quantification of real-time polymerase chain reaction data, the same HKGs should be selected for all groups, we believe that GAPDH and ACTB are suitable HKGs for studies on the effect of hypoxia on cultured kidney cells.

  5. Coculture with endothelial cells enhances osteogenic differentiation of periodontal ligament stem cells via cyclooxygenase-2/prostaglandin E2/vascular endothelial growth factor signaling under hypoxia.

    Science.gov (United States)

    Zhao, Lixing; Wu, Yeke; Tan, Lijun; Xu, Zhenrui; Wang, Jun; Zhao, Zhihe; Li, Xiaoyu; Li, Yu; Yang, Pu; Tang, Tian

    2013-12-01

    During periodontitis and orthodontic tooth movement, periodontal vasculature is severely impaired, leading to a hypoxic microenvironment of periodontal cells. However, the impact of hypoxia on periodontal cells is poorly defined. The present study investigates responses of cocultured endothelial cells (ECs) and periodontal ligament stem cells (PDLSCs) to hypoxia. Osteogenic differentiation, molecular characterization, and various behaviors of PDLSCs and human umbilical venous ECs under hypoxia were assessed by quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Moreover, the effect of ECs on PDLSC osteogenic differentiation was tested using NS398 (cyclooxygenase 2 blocker), SU5416 (vascular endothelial growth factor [VEGF] receptor inhibitor), AH6809, L-798106, and L-161982 (EP1/2/3/4 antagonists). First, hypoxia promoted osteogenic differentiation in PDLSCs and enhanced EC migration, whereas PD98059 (extracellular signal-regulated protein kinase [ERK] inhibitor) blocked, and cocultured ECs further enhanced, hypoxia-induced osteogenic differentiation. Second, NS398 impaired EC migration and prostaglandin E2 (PGE2)/VEGF release, whereas cocultured PDLSCs and exogenous PGE2 partially reversed it. Third, NS398 (pretreated ECs) decreased PGE2/VEGF concentrations. NS398-treated ECs and AH6809/SU5416-treated PDLSCs impaired cocultured EC-induced enhancement of PDLSC osteogenic differentiation. Hypoxia enhances ERK-mediated osteogenic differentiation in PDLSCs. Coculture with EC further augments PDLSC osteogenic differentiation via cyclooxygenase-2/PGE2/VEGF signaling.

  6. Hydrogen sulfide is expressed in the human and the rat cultured nucleus pulposus cells and suppresses apoptosis induced by hypoxia.

    Directory of Open Access Journals (Sweden)

    Haolin Sun

    Full Text Available Apoptosis plays pivotal role in the pathogenesis of degenerative disc diseases, which is the primary contributor to low back pain. Although the role of hydrogen sulfide (H2S in cell apoptosis is well appreciated, the effects and mechanism that H2S regulates the program death of intervertebral disc cell are not yet elucidated. In this study, we utilized the nucleus pulposus (NP from patients with lumbar disc herniation to investigate the relationship between endogenous H2S and NP cells apoptosis in human. Furthermore, we analyzed primary rat NP cells to study the effects of exogenous H2S on hypoxia induced cell apoptosis. Human NP samples were obtained from patients with lumbar disc herniation and were divided into uncontained and contained herniation groups. Using immunohistochemistry staining and sulphur-sensitive electrode, we detected the expression of cystathionine-β-synthase (CBS and cystathionine γ-lyase (CSE, as well as the production of endogenous H2S in human NP. Tunel staining showed increased apoptosis in NP from herniated disc; and there was significant correlation between H2S generation and apoptosis in human NP. CoCl2 was then used to induce hypoxia in cultured primary rat NP cells. Annexin V staining indicated that exogenous NaHS attenuated hypoxia induced apoptosis in rat NP cells. Furthermore, hypoxia significantly increased the levels of multiple apoptosis associated proteins (Fas, Cytochromes C, Caspase 9 and cleaved-Caspase-3 in cells, which were eliminated by NaHS. Our study demonstrates the presence of endogenous H2S in human intervertebral disc; and the endogenous H2S generation rate is associated with NP apoptosis in herniated disc. In vitro study showes exogenous H2S donor attenuates hypoxia induced apoptosis in primary rat NP cells. Thus, our work provides insights that H2S may have beneficial effects in treating degenerative disc diseases.

  7. CD146+ human umbilical cord perivascular cells maintain stemness under hypoxia and as a cell source for skeletal regeneration.

    Directory of Open Access Journals (Sweden)

    Wing Pui Tsang

    Full Text Available The human umbilical cord perivascular cells (HUCPVCs have been considered as an alternative source of mesenchymal progenitors for cell based regenerative medicine. However, the biological properties of these cells remain to be well characterized. In the present study, HUCPVCs were isolated and sorted by CD146(+ pericyte marker. The purified CD146(+ HUCPVCs were induced to differentiate efficiently into osteoblast, chondrocyte and adipocyte lineages in vitro. Six weeks following subcutaneous transplantation of CD146(+ HUCPVCs-Gelfoam-alginate 3D complexes in severe combined immunodeficiency (SCID mice, newly formed bone matrix with embedded osteocytes of donor origin was observed. The functional engraftment of CD146(+ HUCPVCs in the new bone regenerates was further confirmed in a critical-sized bone defect model in SCID mice. Hypoxic conditions suppressed osteogenic differentiation while increased cell proliferation and colony-forming efficiency of CD146(+ HUCPVCs as compared to that under normoxic conditions. Re-oxygenation restored the multi-differentiation potential of the CD146(+ HUCPVCs. Western blot analysis revealed an upregulation of HIF-1α, HIF-2α, and OCT-4 protein expression in CD146(+ HUCPVCs under hypoxia, while there was no remarkable change in SOX2 and NANOG expression. The gene expression profiles of stem cell transcription factors between cells treated by normoxia and hypoxic conditions were compared by PCR array analysis. Intriguingly, PPAR-γ was dramatically downregulated (20-fold in mRNA expression under hypoxia, and was revealed to possess a putative binding site in the Hif-2α gene promoter region. Chromatin immunoprecipitation assays confirmed the binding of PPAR-γ protein to the Hif-2α promoter and the binding was suppressed by hypoxia treatment. Luciferase reporter assay showed that the Hif-2α promoter activity was suppressed by PPAR expression. Thus, PPAR-γ may involve in the regulation of HIF-2α for stemness

  8. A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

    DEFF Research Database (Denmark)

    Greenberger, Lee M; Horak, Ivan D; Filpula, David

    2008-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth factor...... the expression of HIF-1alpha mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC(50), 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor......-regulation of endogenous HIF-1alpha and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted...

  9. Nitroimidazoles as hypoxic cell radiosensitizers and hypoxia probes: misonidazole, myths and mistakes.

    Science.gov (United States)

    Wardman, Peter

    2018-03-20

    Nitroimidazoles have been extensively explored as hypoxic cell radiosensitizers but have had limited clinical success, with efficacy restricted by toxicity. However, they have proven clinically useful as probes for tumour hypoxia. Both applications, and probably much of the dose-limiting toxicities, reflect the dominant chemical property of electron affinity or ease of reduction, associated with the nitro substituent in an aromatic structure. This single dominant property affords unusual, indeed extraordinary flexibility in drug or probe design, suggesting further development is possible in spite of earlier limitations, in particular building on the benefit of hindsight and an appreciation of errors made in earlier studies. The most notable errors were: the delay in viewing cellular thiol depletion as a likely common artefact in testing in vitro; slow recognition of pH-driven concentration gradients when compounds were weak acids and bases; and a failure to explore the possible involvement of pH and ascorbate in influencing hypoxia probe binding. The experience points to the need to involve a wider range of expertise than that historically involved in many laboratories when studying the effects of chemicals on radiation response or using diagnostic probes.

  10. Abnormal autonomic cardiac response to transient hypoxia in sickle cell anemia

    International Nuclear Information System (INIS)

    Sangkatumvong, S; Khoo, M C K; Coates, T D

    2008-01-01

    The objective of this study was to non-invasively assess cardiac autonomic control in subjects with sickle cell anemia (SCA) by tracking the changes in heart rate variability (HRV) that occur following brief exposure to a hypoxic stimulus. Five African–American SCA patients and seven healthy control subjects were recruited to participate in this study. Each subject was exposed to a controlled hypoxic stimulus consisting of five breaths of nitrogen. Time-varying spectral analysis of HRV was applied to estimate the cardiac autonomic response to the transient episode of hypoxia. The confounding effects of changes in respiration on the HRV spectral indices were reduced by using a computational model. A significant decrease in the parameters related to parasympathetic control was detected in the post-hypoxic responses of the SCA subjects relative to normal controls. The spectral index related to sympathetic activity, on the other hand, showed a tendency to increase the following hypoxic stimulation, but the change was not significant. This study suggests that there is some degree of cardiovascular autonomic dysfunction in SCA that is revealed by the response to transient hypoxia

  11. Hypoxia induces a phase transition within a kinase signaling network in cancer cells.

    Science.gov (United States)

    Wei, Wei; Shi, Qihui; Remacle, Francoise; Qin, Lidong; Shackelford, David B; Shin, Young Shik; Mischel, Paul S; Levine, R D; Heath, James R

    2013-04-09

    Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)--a critical component of hypoxic signaling and a compelling cancer drug target--is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier's principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles.

  12. Hypoxia induces a phase transition within a kinase signaling network in cancer cells

    Science.gov (United States)

    Wei, Wei; Shi, Qihui; Remacle, Francoise; Qin, Lidong; Shackelford, David B.; Shin, Young Shik; Mischel, Paul S.; Levine, R. D.; Heath, James R.

    2013-01-01

    Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)—a critical component of hypoxic signaling and a compelling cancer drug target—is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier’s principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles. PMID:23530221

  13. Hypoxia-induced suppression of c-Myc by HIF-2α in human pulmonary endothelial cells attenuates TFAM expression.

    Science.gov (United States)

    Zarrabi, Ali J; Kao, Derrick; Nguyen, Dustin T; Loscalzo, Joseph; Handy, Diane E

    2017-10-01

    The adaptive response to hypoxia is mediated in large part by stabilization of the hypoxia-inducible factors, HIF-1α and HIF-2α. A hallmark of this response is the metabolic shift to decreased oxidative phosphorylation and increased glycolysis. We hypothesized that hypoxic responses would include a suppression of mitochondrial gene expression. We determined the effects of hypoxia on TFAM, a key mitochondrial transcription factor, in normal pulmonary artery endothelial cells. Hypoxia decreased gene expression of TFAM and that of its upstream regulator, the transcriptional co-activator PGC1β. Although HIF-1α and HIF-2α pathways both contributed to hypoxia-mediated PGC1β suppression, TFAM suppression was regulated solely by HIF-2α-dependent mechanisms. We found that HIF-2α suppresses TFAM by decreasing c-Myc expression. In addition, we show a role for c-Jun in this pathway, linking HIF-2α with attenuation of c-Jun activation. Taken together, these findings establish a new link between HIF-2α and MAPK-signaling that mediates the adaptive regulation of mitochondrial gene expression under low oxygen tension. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    Kroening, Sven; Neubauer, Emily; Wullich, Bernd; Aten, Jan; Goppelt-Struebe, Margarete

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009;

  15. Andrographolide inhibits hypoxia-induced hypoxia-inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP-5 pathways in EA.hy926 cells.

    Science.gov (United States)

    Lin, Hung-Chih; Su, Shih-Li; Lin, Wan-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Lii, Chong-Kuei; Chen, Haw-Wen

    2018-03-01

    Andrographolide is a potent anti-inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET-1) is an endothelium-derived vasoconstrictor with pro-inflammatory properties secreted in response to hypoxia. Mitogen-activated protein kinase phosphatase 5 (MKP-5) is a dual-specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia-induced HIF-1α expression and ET-1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP-5 plays in andrographolide's inhibition of hypoxia-induced expression of HIF-1α and ET-1. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2 . Andrographolide enhanced HO-1 and MKP-5 expression and cellular cGMP content in addition to inhibiting hypoxia-induced ROS generation. Concomitantly, the HO-1 byproduct CO and the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) increased MKP-5 expression, and pretreatment with CO and 8-Br-cGMP inhibited hypoxia-induced HIF-1α and ET-1 expression. Transfection of HO-1 siRNA or pretreatment with the HO-1 inhibitor ZnPP-9 or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide-induced MKP-5 expression. Moreover, silencing MKP-5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia-induced p38 MAPK activation and HIF-1α expression. The inhibition of hypoxia-induced HIF-1α and ET-1 expression by andrographolide is likely associated with HO-1/CO/cGMP/MKP-5 pathways, which is involved in inhibiting hypoxia-induced p38 MAPK activation. © 2017 Wiley Periodicals, Inc.

  16. Mxd1 mediates hypoxia-induced cisplatin resistance in osteosarcoma cells by repression of the PTEN tumor suppressor gene.

    Science.gov (United States)

    Zheng, Datong; Wu, Weiling; Dong, Na; Jiang, Xiuqin; Xu, Jinjin; Zhan, Xi; Zhang, Zhengdong; Hu, Zhenzhen

    2017-10-01

    Hypoxia-induced chemoresistance remains a major obstacle to treating osteosarcoma effectively. Mxd1, a member of the Myc/Max/Mxd family, was shown to be involved in the development of drug resistance under hypoxia. However, the effect of Mxd1 on hypoxia-induced cisplatin (CDDP) resistance and its mechanism in osteosarcoma have not been fully elucidated. In this study, we demonstrated that HIF-1α-induced Mxd1 contributed to CDDP resistance in hypoxic U-2OS and MG-63 cells. The knockdown of Mxd1 expression elevated PTEN expression at both protein and RNA levels in these hypoxic cells. Using Luciferase reporter and ChIP assays, we confirmed that Mxd1 directly bound to the E-box sites within the PTEN promoter region. We further demonstrated that PTEN knockdown decreased CDDP sensitivity in Mxd1 siRNA-transfected U-2OS and MG-63 cells under hypoxia. Our results also showed that Mxd1 deficiency in hypoxic U-2OS and MG-63 cells lead to inactivation of PI3K/AKT signaling, which is the downstream of PTEN. Furthermore, blockade of PI3K/AKT signal re-sensitized hypoxic U-2OS and MG-63 cells to CDDP. Taken together, these findings suggest that HIF-1α-induced Mxd1 up-regulation suppresses the expression of PTEN under hypoxia, which leads to the activation of PI3K/AKT antiapoptotic and survival pathway. As a result CDDP resistance in osteosarcoma cells is induced. © 2017 Wiley Periodicals, Inc.

  17. Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells

    Directory of Open Access Journals (Sweden)

    O. H. Minchenko

    2016-06-01

    Full Text Available We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1, which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2, malic enzyme 2 (ME2, mitochondrial aspartate aminotransferase (GOT2, and subunit B of succinate dehydrogenase (SDHB in control (transfected by empty vector glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2 and subunit D of succinate dehydrogenase (SDHD genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

  18. Transcriptome sequencing revealed differences in the response of renal cancer cells to hypoxia and CoCl2 treatment [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Nadezhda Zhigalova

    2015-12-01

    Full Text Available Human cancer cells are subjected to hypoxic conditions in many tumours. Hypoxia causes alterations in the glycolytic pathway activation through stabilization of hypoxia-inducible factor 1. Currently, two approaches are commonly used to model hypoxia: an alternative to generating low-oxygen conditions in an incubator, cells can be treated with CoCl2. We performed RNA-seq experiments to study transcriptomes of human Caki-1 cells under real hypoxia and after CoCl2 treatment. Despite causing transcriptional changes of a much higher order of magnitude for the genes in the hypoxia regulation pathway, CoCl2 treatment fails to induce alterations in the glycolysis / gluconeogenesis pathway. Moreover, CoCl2 caused aberrant activation of other oxidoreductases in glycine, serine and threonine metabolism pathways.

  19. Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Kučera, Jan; Netušilová, Julie; Sladeček, Stanislava; Lánová, Martina; Vašíček, Ondřej; Štefková, Kateřina; Navrátilová, Jarmila; Kubala, Lukáš; Pacherník, Jiří

    2017-01-01

    Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1 α -deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O 2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.

  20. Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Jan Kučera

    2017-01-01

    Full Text Available Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1 is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.

  1. Exogenous pyruvate facilitates cancer cell adaptation to hypoxia by serving as an oxygen surrogate.

    Science.gov (United States)

    Yin, Chengqian; He, Dan; Chen, Shuyang; Tan, Xiaoling; Sang, Nianli

    2016-07-26

    Molecular oxygen is the final electron acceptor in cellular metabolism but cancer cells often become adaptive to hypoxia, which promotes resistance to chemotherapy and radiation. The reduction of endogenous glycolytic pyruvate to lactate is known as an adaptive strategy for hypoxic cells. Whether exogenous pyruvate is required for hypoxic cell proliferation by either serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxic and ρ0 cells defective in electron transfer chain, we show that exogenous pyruvate is required to sustain proliferation of both cancer and non-cancer cells that cannot utilize oxygen. Particularly, we show that absence of pyruvate led to glycolysis inhibition and AMPK activation along with decreased NAD+ levels in ρ0 cells; and exogenous pyruvate increases lactate yield, elevates NAD+/NADH ratio and suppresses AMPK activation. Knockdown of lactate dehydrogenase significantly inhibits the rescuing effects of exogenous pyruvate. In contrast, none of pyruvate-derived metabolites tested (including acetyl-CoA, α-ketoglutarate, succinate and alanine) can replace pyruvate in supporting ρ0 cell proliferation. Knockdown of pyruvate carboxylase, pyruvate dehydrogenase and citrate synthase do not impair exogenous pyruvate to rescue ρ0 cells. Importantly, we show that exogenous pyruvate relieves ATP insufficiency and mTOR inhibition and promotes proliferation of hypoxic cells, and that well-oxygenated cells release pyruvate, providing a potential in vivo source of pyruvate. Taken together, our data support a novel pyruvate cycle model in which oxygenated cells release pyruvate for hypoxic cells as an oxygen surrogate. The pyruvate cycle may be targeted as a new therapy of hypoxic cancers.

  2. Hypoxia-inducible factor-1α perpetuates synovial fibroblast interactions with T cells and B cells in rheumatoid arthritis.

    Science.gov (United States)

    Hu, Fanlei; Liu, Hongjiang; Xu, Liling; Li, Yingni; Liu, Xu; Shi, Lianjie; Su, Yin; Qiu, Xiaoyan; Zhang, Xia; Yang, Yuqin; Zhang, Jian; Li, Zhanguo

    2016-03-01

    Synovial fibroblast hyperplasia, T-cell hyperactivity, B-cell overactivation, and the self-perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia-inducible factor-1α (HIF-1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF-1α regulates interactions between RASFs and T cells and B cells. We report here that HIF-1α promotes the expression of inflammatory cytokines IL-6, IL-8, TNF-α, and IL-1β, and cell-cell contact mediators IL-15, vascular cell adhesion molecule (VCAM)-1, thrombospondin (TSP)-1, and stromal cell-derived factor (SDF)-1 in RASFs. Furthermore, HIF-1α perpetuates RASF-mediated inflammatory Th1- and Th17-cell expansion while differentially inhibiting regulatory B10 and innate-like B cells, leading to increased IFN-γ, IL-17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF-1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF-1α may provide new therapeutic strategies for overcoming this persistent disease. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Proteomic Analysis and Identification of Paracrine Factors in Mesenchymal Stem Cell-Conditioned Media under Hypoxia

    Directory of Open Access Journals (Sweden)

    Suk-Won Song

    2016-11-01

    Full Text Available Background/Aims: We previously showed that a hypoxic environment modulates the antiarrhythmic potential of mesenchymal stem cells. Methods: To investigate the mechanism by which secreted proteins contribute to the pathogenesis of antiarrhythmic potential in mesenchymal stem cells, we used two-dimensional electrophoresis combined with MALDI-TOF-MS to perform a proteomic analysis to compare the paracrine media produced by normoxic and hypoxic cells. Results: The proteomic analysis revealed that 66 protein spots out of a total of 231 matched spots indicated differential expression between the normoxic and hypoxic conditioned media of mesenchymal stem cells. Interestingly, two tropomyosin isoforms were dramatically increased in the hypoxic conditioned medium of mesenchymal stem cells. An increase in tropomyosin was confirmed using Western blot to analyze the conditioned media between normoxic and hypoxic cells. In a network analysis based on gene ontology (GO Molecular Function by GeneMANIA analysis, most of the identified proteins were found to be involved in the regulation of heart processes. Conclusion: Our results show that hypoxia up-regulates tropomyosin and other secreted proteins which suggests that tropomyosin may be involved in regulating proarrhythmic and antiarrhythmic functions.

  4. Hypoxia and Mucosal Inflammation

    Science.gov (United States)

    Colgan, Sean P.; Campbell, Eric L.; Kominsky, Douglas J.

    2016-01-01

    Sites of inflammation are defined by significant changes in metabolic activity. Recent studies have suggested that O2 metabolism and hypoxia play a prominent role in inflammation so-called “inflammatory hypoxia,” which results from a combination of recruited inflammatory cells (e.g., neutrophils and monocytes), the local proliferation of multiple cell types, and the activation of multiple O2-consuming enzymes during inflammation. These shifts in energy supply and demand result in localized regions of hypoxia and have revealed the important function off the transcription factor HIF (hypoxia-inducible factor) in the regulation of key target genes that promote inflammatory resolution. Analysis of these pathways has provided multiple opportunities for understanding basic mechanisms of inflammation and has defined new targets for intervention. Here, we review recent work addressing tissue hypoxia and metabolic control of inflammation and immunity. PMID:27193451

  5. Hypoxia. 3. Hypoxia and neurotransmitter synthesis

    OpenAIRE

    Kumar, Ganesh K.

    2011-01-01

    Central and peripheral neurons as well as neuroendocrine cells express a variety of neurotransmitters/modulators that play critical roles in regulation of physiological systems. The synthesis of several neurotransmitters/modulators is regulated by O2-requiring rate-limiting enzymes. Consequently, hypoxia resulting from perturbations in O2 homeostasis can affect neuronal functions by altering neurotransmitter synthesis. Two broad categories of hypoxia are frequently encountered: continuous hyp...

  6. Hydrogen Suppresses Hypoxia/Reoxygenation-Induced Cell Death in Hippocampal Neurons Through Reducing Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Rong Wei

    2015-05-01

    Full Text Available Background & Aims: Deep hypothermic circulatory arrest (DHCA is a cerebral protection technique that has been used in the operations involving the aortic arch and brain aneurysm for decades. We previous showed that DHCA treated rats developed a significant oxidative stress and apoptosis in neurons. We here intend to investigate the protective the effect of hydrogen against oxidative stress-induced cell injury and the involved mechanisms using an in vitro experimental model of hypoxia/reoxygenation (H/R on HT-22 cells. Methods: The model of H/R was established using an airtight culture container and the anaeropack. Measurement of mitochondrial membrane potential (MMP and reactive oxygen species (ROS production was used H2DCFDA and JC-1 staining. Western blot was used for the quantification of Akt, p-Akt, Bcl-2, Bax and cleaved caspase-3 proteins. The microRNA (miRNA profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing. Results: The elevation of ROS and reduction of MMP were significantly induced by the treatment with hypoxia for 18 h followed by reoxygenation for 6 h. Hydrogen treatment significantly reduced H/R-caused cell death. The levels of p-Akt (Ser 473 and Bcl-2 were significantly increased while Bax and cleaved caspase-3 were decreased by hydrogen treatment on the model of H/R. The expression of miR-200 family was significantly elevated in model of DHCA and H/R. Hydrogen administration inhibited the H/R-induced expression of miR-200 family in HT-22 cells. In addition, inhibition of miR-200 family suppressed H/R-caused cell death through reducing ROS production. Conclusions: These results suggest that H/R causes oxidative stress-induced cell death and that the hydrogen protects against H/R-induced cell death in HT22 cells, in part, due to reducing expression of miR-200 family.

  7. CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1α

    International Nuclear Information System (INIS)

    Esencay, Mine; Sarfraz, Yasmeen; Zagzag, David

    2013-01-01

    Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion. In order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test. In this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7. Taken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors

  8. Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2

    Directory of Open Access Journals (Sweden)

    Jung-Seok Lee

    2018-04-01

    Full Text Available This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2 treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis, and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Keywords: Bone marrow stromal cells, Hypoxia, Fibroblast growth factor, Tissue regeneration, Microenvironment interactions

  9. Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification.

    Science.gov (United States)

    Mokas, Sophie; Larivière, Richard; Lamalice, Laurent; Gobeil, Stéphane; Cornfield, David N; Agharazii, Mohsen; Richard, Darren E

    2016-09-01

    Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  10. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2016-01-01

    Full Text Available Heat shock protein 70 (HSP70 maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R, thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  11. Effects of hypoxia on expression of a panel of stem cell and chemosensitivity markers in glioblastoma cell line-derived spheroids

    DEFF Research Database (Denmark)

    Kolenda, Jesper; Jensen, Stine Skov; Aaberg-Jessen, Charlotte

    Glioblastomas are the most frequent and malignant primary brain tumor. Tumor stem cells in these tumors have recently been suggested to possess innate resistance mechanisms against radiation and chemotherapy possibly explaining their high level of therapeutic resistance. Moreover tumor hypoxia...... of a panel of stem cell and chemosensitivity markers was therefore investigated using glioma spheroids derived from the conventional glioblastoma cell line U87. The glioma spheroids were derived at normoxic (21% O2) and hypoxic (1% O2) culturing conditions in serum-free medium with EGF and bFGF. The entire...... immunohistochemical panel included hypoxia (HIF-1α, HIF-2α), proliferation (Ki-67) and stem cell (CD133, nestin, podoplanin, Bmi-1, Sox-2) markers as well as markers related to chemosensitivity (MGMT, MDR-1, TIMP-1, Lamp-1). Since spheroids derived in hypoxia were smaller than in normoxia, a set of experiments...

  12. Hypoxia and inflammation in children with sickle cell disease: implications for hippocampal functioning and episodic memory.

    Science.gov (United States)

    Iampietro, Mary; Giovannetti, Tania; Tarazi, Reem

    2014-06-01

    Children with sickle cell disease (SCD) suffer from systemic processes (e.g., chronic anemia, recurrent hypoxic-ischemic events, chronic inflammation) that have been associated with neurocognitive impairment in a range of clinical populations, but which have been largely understudied in relation to specific domains of cognitive functioning in children with SCD. This review focuses on episodic memory, as the hippocampus may be especially vulnerable to the systemic processes associated with SCD. The first part of the paper outlines the pathophysiology of SCD and briefly reviews the extant literature on academic and cognitive functioning in children with SCD, emphasizing the dearth of research on episodic memory. Next, the complex systemic processes of hypoxia and inflammation associated with SCD are reviewed, along with research that has associated these processes with hippocampal damage and memory impairment. The paper concludes with suggestions for future research that are informed, in part, by the literature on developmental amnesia.

  13. Dataset on the activation of Müller cells through macrophages upon hypoxia in the retina

    Directory of Open Access Journals (Sweden)

    Christina Nürnberg

    2018-02-01

    Full Text Available The dataset presented in this article complements the article entitled “Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells” (C. Nürnberg, N. Kociok, C. Brockmann, T. Lischke, S. Crespo-Garcia, N. Reichhart, S. Wolf, R. Baumgrass, S.A. Eming, S. Beer-Hammer, and A.M. Joussen. This complementary dataset provides further insight into the experimental validation of the VEGFfl/fl LysMCre (here named VEGFmcko knockout model used in the main article through genomic and quantitative Real-Time PCR in various murine tissues as well as additional flow cytometry data and immunohistochemical stainings. By providing these data, we aim to enable researcher to reproduce and critically analyze our data.

  14. Hypoxia. Cross talk between oxygen sensing and the cell cycle machinery.

    Science.gov (United States)

    Semenza, Gregg L

    2011-09-01

    A fundamental physiological property of mammalian cells is the regulation of proliferation according to O(2) availability. Progression through the cell cycle is inhibited under hypoxic conditions in many, but not all, cell types, and this G1 arrest is dependent on hypoxia-inducible factor (HIF) 1α. Components of the hexameric MCM helicase, which binds to replication origins before the onset of DNA synthesis, are present in large excess in mammalian cells relative to origins, suggesting that they may have additional functions. Screens for HIF-1α interacting proteins revealed that MCM7 binds to the amino-terminal PER-SIM-ARNT (PAS) domain of HIF-1α and stimulates prolyl hydroxylation-dependent ubiquitination and degradation of HIF-1α, whereas MCM3 binds to the carboxyl terminus of HIF-1α and enhances asparaginyl hydroxylation-dependent inhibition of HIF-1α transactivation domain function. Thus MCM proteins inhibit HIF activity via two distinct O(2)-dependent mechanisms. Under prolonged hypoxic conditions, MCM mRNA expression is inhibited in a HIF-1α-dependent manner. Thus HIF and MCM proteins act in a mutually antagonistic manner, providing a novel molecular mechanism for homeostatic regulation of cell proliferation based on the relative levels of these proteins.

  15. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Grayson, Warren L.; Zhao, Feng; Bunnell, Bruce; Ma, Teng

    2007-01-01

    Changes in oxygen concentrations affect many of the innate characteristics of stem and progenitor cells. Human mesenchymal stem cells (hMSCs) were maintained under hypoxic atmospheres (2% O 2 ) for up to seven in vitro passages. This resulted in approximately 30-fold higher hMSC expansion over 6 weeks without loss of multi-lineage differentiation capabilities. Under hypoxia, hMSCs maintained their growth-rates even after reaching confluence, resulting in the formation of multiple cell layers. Hypoxic hMSCs also displayed differences in the cell and nuclear morphologies as well as enhanced ECM formation and organization. These changes in cellular characteristics were accompanied by higher mRNA levels of Oct-4 and HIF-2α, as well as increased expression levels of connexin-43, a protein used in gap junction formation. The results from this study demonstrated that oxygen concentrations affected many aspects of stem-cell physiology, including growth and in vitro development, and may be a critical parameter during expansion and differentiation

  16. CoCl2, a mimic of hypoxia, induces formation of polyploid giant cells with stem characteristics in colon cancer.

    Directory of Open Access Journals (Sweden)

    Laura M Lopez-Sánchez

    Full Text Available The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl2 induced the formation of cells with larger cell and nuclear size (PGCCs, while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl2 treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl2 treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47 and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.

  17. Hypoxia-induced PD-L1/PD-1 crosstalk impairs T-cell function in sleep apnoea.

    Science.gov (United States)

    Cubillos-Zapata, Carolina; Avendaño-Ortiz, Jose; Hernandez-Jimenez, Enrique; Toledano, Victor; Casas-Martin, Jose; Varela-Serrano, Anibal; Torres, Marta; Almendros, Isaac; Casitas, Raquel; Fernández-Navarro, Isabel; Garcia-Sanchez, Aldara; Aguirre, Luis A; Farre, Ramón; López-Collazo, Eduardo; García-Rio, Francisco

    2017-10-01

    Obstructive sleep apnoea (OSA) is associated with higher cancer incidence, tumour aggressiveness and cancer mortality, as well as greater severity of infections, which have been attributed to an immune deregulation. We studied the expression of programmed cell death (PD)-1 receptor and its ligand (PD-L1) on immune cells from patients with OSA, and its consequences on immune-suppressing activity. We report that PD-L1 was overexpressed on monocytes and PD-1 was overexpressed on CD8 + T-cells in a severity-dependent manner. PD-L1 and PD-1 overexpression were induced in both the human in vitro and murine models of intermittent hypoxia, as well as by hypoxia-inducible factor-1α transfection. PD-L1/PD-1 crosstalk suppressed T-cell proliferation and activation of autologous T-lymphocytes and impaired the cytotoxic activity of CD8 + T-cells. In addition, monocytes from patients with OSA exhibited high levels of retinoic acid related orphan receptor, which might explain the differentiation of myeloid-derived suppressor cells. Intermittent hypoxia upregulated the PD-L1/PD-1 crosstalk in patients with OSA, resulting in a reduction in CD8 + T-cell activation and cytotoxicity, providing biological plausibility to the increased incidence and aggressiveness of cancer and the higher risk of infections described in these patients. Copyright ©ERS 2017.

  18. Suitable housekeeping genes for normalization of transcript abundance analysis by real-time RT-PCR in cultured bovine granulosa cells during hypoxia and differential cell plating density.

    Science.gov (United States)

    Baddela, Vijay S; Baufeld, Anja; Yenuganti, Vengala R; Vanselow, Jens; Singh, Dheer

    2014-11-27

    Bovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions. During the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard. Three of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed. The present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.

  19. Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells

    International Nuclear Information System (INIS)

    Staab, Adrian; Einsele, Hermann; Flentje, Michael; Vordermark, Dirk; Loeffler, Jürgen; Said, Harun M; Diehlmann, Désirée; Katzer, Astrid; Beyer, Melanie; Fleischer, Markus; Schwab, Franz; Baier, Kurt

    2007-01-01

    Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells. Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O 2 , 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h). Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 ± 7.2% and 39.6 ± 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively. HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

  20. Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells

    Directory of Open Access Journals (Sweden)

    Baier Kurt

    2007-11-01

    Full Text Available Abstract Background Hypoxia-inducible factor-1 (HIF-1 overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells. Methods Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9 and vascular endothelial growth factor (VEGF by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O2, 12 h conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h. Results Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 ± 7.2% and 39.6 ± 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER' compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively. Conclusion HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

  1. Hypoxia preconditioned mesenchymal stem cells prevent cardiac fibroblast activation and collagen production via leptin.

    Directory of Open Access Journals (Sweden)

    Panpan Chen

    Full Text Available Activation of cardiac fibroblasts into myofibroblasts constitutes a key step in cardiac remodeling after myocardial infarction (MI, due to interstitial fibrosis. Mesenchymal stem cells (MSCs have been shown to improve post-MI remodeling an effect that is enhanced by hypoxia preconditioning (HPC. Leptin has been shown to promote cardiac fibrosis. The expression of leptin is significantly increased in MSCs after HPC but it is unknown whether leptin contributes to MSC therapy or the fibrosis process. The objective of this study was to determine whether leptin secreted from MSCs modulates cardiac fibrosis.Cardiac fibroblast (CF activation was induced by hypoxia (0.5% O2. The effects of MSCs on fibroblast activation were analyzed by co-culturing MSCs with CFs, and detecting the expression of α-SMA, SM22α, and collagen IαI in CFs by western blot, immunofluorescence and Sirius red staining. In vivo MSCs antifibrotic effects on left ventricular remodeling were investigated using an acute MI model involving permanent ligation of the left anterior descending coronary artery.Co-cultured MSCs decreased fibroblast activation and HPC enhanced the effects. Leptin deficit MSCs from Ob/Ob mice did not decrease fibroblast activation. Consistent with this, H-MSCs significantly inhibited cardiac fibrosis after MI and mediated decreased expression of TGF-β/Smad2 and MRTF-A in CFs. These effects were again absent in leptin-deficient MSCs.Our data demonstrate that activation of cardiac fibroblast was inhibited by MSCs in a manner that was leptin-dependent. The mechanism may involve blocking TGF-β/Smad2 and MRTF-A signal pathways.

  2. Hypoxia Room

    Data.gov (United States)

    Federal Laboratory Consortium — The Hypoxia Room is a 8x8x8 ft. clear vinyl plastic and aluminum frame construction enclosure located within USAREIM laboratory 028. The Hypoxia Room (manufactured...

  3. Au@Pt nanoparticles as catalase mimics to attenuate tumor hypoxia and enhance immune cell-mediated cytotoxicity

    Science.gov (United States)

    Liang, Hong; Wu, Ying; Ou, Xiang-Yu; Li, Jing-Ying; Li, Juan

    2017-11-01

    Hypoxic tumor microenvironment (TME) is closely linked to tumor progression, heterogeneity and immune suppression. Therefore, the development of effective methods to overcome hypoxia and substantially enhance the immunotherapy efficacy remains a desirable goal. Herein, we engineered a biocompatible Au core/Pt shell nanoparticles (Au@Pt NPs) to reoxygenate the TME by reacting with endogenous H2O2. Treatment with Au@Pt NPs appeared to improve oxygen in intracellular environments and decrease hypoxia-inducible factor-1α expression. Furthermore, the integration of high catalytic efficiency of Au@Pt NPs with cytokine-induced killer (CIK) cell immunotherapy, could lead to significantly improve the effect of CIK cell-mediated cytotoxicity. These results suggest great potential of Au@Pt NPs for regulation of the hypoxic TME and enhance immune cell mediated anti-tumor immunity.

  4. Proteomic Alterations in Response to Hypoxia Inducible Factor 2α in Normoxic Neuroblastoma Cells.

    Science.gov (United States)

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Persano, Luca; Vitale, Monica; Sassi, Mauro; Bresolin, Silvia; Serafin, Valentina; Zambrano, Nicola; Scaloni, Andrea; Basso, Giuseppe; Iolascon, Achille; Capasso, Mario

    2016-10-07

    Hypoxia inducible factor (HIF)-2α protein expression in solid tumors promotes stem-like phenotype in cancer stem cells and increases tumorigenic potential in nonstem cancer cells. Recently, we have shown that HIF-1/2α gene expression is correlated to neuroblastoma (NB) poor survival and to undifferentiated tumor state; HIF-2α protein was demonstrated to enhance aggressive features of the disease. In this study, we used proteomic experiments on NB cells to investigate HIF-2α downstream-regulated proteins or pathways with the aim of providing novel therapeutic targets or bad prognosis markers. We verified that pathways mostly altered by HIF-2α perturbation are involved in tumor progression. In particular, HIF-2α induces alteration of central metabolism and splicing control pathways. Simultaneously, WNT, RAS/MAPK, and PI3K/AKT activity or expression are affected and may impact the sensitivity and the intensity of HIF-2α-regulated pathways. Furthermore, genes coding the identified HIF-2α-related markers built a signature able to stratify NB patients with unfavorable outcome. Taken together, our findings underline the relevance of dissecting the downstream effects of a poor survival marker in developing targeted therapy and improving patient stratification. Future prospective studies are needed to translate the use of these data into the clinical practice.

  5. Identification of agents that reduce renal hypoxia-reoxygenation injury using cell-based screening: purine nucleosides are alternative energy sources in LLC-PK1 cells during hypoxia.

    Science.gov (United States)

    Szoleczky, Petra; Módis, Katalin; Nagy, Nóra; Dóri Tóth, Zoltán; DeWitt, Douglas; Szabó, Csaba; Gero, Domokos

    2012-01-01

    Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Hypoxia pretreatment and EPO-modification enhance the protective effects of MSC on neuron-like PC12 cells in a similar way.

    Science.gov (United States)

    Feng, Jinli; Wang, Wei

    2017-01-08

    Mesenchymal stem cells (MSC) based cell transplantation therapy is proved to be an attractive strategy with great potential for improvement of hypoxia induced neural damage. In the present study, MSCs were co-culture with PC12 to investigate its protective effects against hypoxia pretreatment, and the Lactate dehydrogenase (LDH) release assay, MTT and Anexin V staining were performed to analysis the cellular damage or apoptotic. RT-PCR and Western blotting were further used to investigate the underlying mechanism. The results indicate that hypoxia treatment results in the decrease of PC12 cell viability, yet co-culture with MSC could protect the PC12 from hypoxia induced damage. Hypoxia pre-activated or EPO transduced MSC with up-regulated erythropoietin (EPO) expression could further enhance MSC's protective effect against hypoxia induced cell damage, which was associated with high level of anti-apoptotic p-Akt and ration Bcl-2/Bax, and decreased Caspase 3 in PC12. Taken together, these data suggests high levels of MSC-mediated cyto-protection is closely tied to high gene expression levels of EPO. The up-regulation of EPO for enhanced MSC-mediated cyto-protection may has great potential for the MSC cellular therapy of neural or neuronal injuries induced by hypoxia. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. [Influence of Erigeron breviscapus on the expression of collagen type I , MMP1 and TIMP1 of MRC-5 cells under hypoxia].

    Science.gov (United States)

    Zheng, Jia-Qun; Xie, Min; Gao, Yan; Zhang, Xu

    2012-05-01

    Study the influence of Erigeron breviscapus on collagen type I, matrix metalloproteinase1 (MMP1) and tissue inhibitor of metalloproteinase1(TIMP1) in human embryo lung fibroblast MRC-5 under hypoxia. MRC-5 cultured in vitro were individed into 4 groups, including normoxia control, hypoxia, hypoxia and staurosporine (SP), hypoxia and erigeron breviscapus. The expression of hypoxia-inducible factor-1 alpha (HIF-1alpha), pro-collal and phospho-smad2 (p-smad2) were evaluated by Western blot. The level of collagen type I, MMP1 and TIMP1 in supernatant fluid were detected by ELISA. Expressed pro-collalpha1, p-smad2 in MRC-5 cells under nomoxia were detected, but not HIF-lalpha. A low level of collagen type I, MMP1 and TIMP1 was also detected in the cell supernatant. In nomoxia secreted pro-collalpha1, p-smad2, collagen type I , MMP1 and TIMP1, hardly secreted HIF-lalpha. Under hypoxia, the expression of all of them was increased when compared with those of under nomoxia condition (P MRC-5 cells and collagen type I, MMP1, TIMP1 in supernatant fluid were observed (P MRC-5 cells under hypoxia condition by inhibit the expression of collagen type I and TIMP1 partly through the Smad2 signaling pathways.

  8. Hypoxia increases ROS signaling and cytosolic Ca(2+) in pulmonary artery smooth muscle cells of mouse lungs slices.

    Science.gov (United States)

    Desireddi, Jennifer R; Farrow, Kathryn N; Marks, Jeremy D; Waypa, Gregory B; Schumacker, Paul T

    2010-03-01

    Precapillary arteries constrict during alveolar hypoxia in a response known as hypoxic pulmonary vasoconstriction (HPV). The mechanism by which pulmonary arterial smooth muscle cells (PASMCs) detect a decrease in Po(2) and trigger contraction is not fully understood. Previous studies in cultured PASMCs show that hypoxia induces an increase in reactive oxygen species (ROS) production, but these results may not reflect responses of PASMCs in their native tissue environment. We therefore assessed hypoxia-induced changes in cytosolic ROS in PASMCs of precision-cut mouse lung slices expressing the redox-sensitive protein, RoGFP. Superfusion of lung slices with hypoxic media (1.5% O(2)) resulted in a significant oxidation of RoGFP from normoxic baseline that was attenuated by overexpression of cytosolic catalase. Hypoxic superfusion also increased [Ca(2+)](i) above normoxic baseline; this response was significantly attenuated by cytosolic catalase overexpression or by the administration of EUK134, a synthetic SOD-catalase mimetic. The hypoxia-induced increase in [Ca(2+)](i) was abolished in the absence of extracellular Ca(2+), indicating that ROS signals trigger entry of extracellular calcium. Collectively, these results indicate that an increase in cytosolic ROS signaling is required for the increase in [Ca(2+)](i) in PASMCs in precision-cut mouse lung slices during the acute HPV response.

  9. 4-Guanidino-n-butyl syringate (Leonurine, SCM 198) protects H9c2 rat ventricular cells from hypoxia-induced apoptosis.

    Science.gov (United States)

    Liu, Xin-hua; Chen, Pei-fang; Pan, Li-long; Silva, Ranil De; Zhu, Yi-zhun

    2009-11-01

    In the present study, we examined the ability of a chemically synthesized compound based on the structure of leonurine, a phytochemical component of Herba leonuri, to protect H9c2 rat ventricular cells from apoptosis induced by hypoxia and serum deprivation, as a model of ischemia. The results revealed a concentration-dependent increase in cell viability associated with leonurine treatment, accompanied by a consistent decline in lactate dehydrogenase leakage into the culture medium. The fraction of annexin V-fluorescein isothiocyanate-positive cells was increased by hypoxia but reduced by leonurine. These changes were associated with increased expression of the antiapoptotic gene, Bcl-2, and reduced expression of the proapoptotic gene, Bax. Leonurine also reduced the cytosolic Ca overload induced by hypoxia. These results suggest that leonurine elicits potent cardioprotective effects in H9c2 cells, and these effects may be mediated by inhibition of intracellular Ca overload and apoptosis during hypoxia.

  10. Folic Acid Represses Hypoxia-Induced Inflammation in THP-1 Cells through Inhibition of the PI3K/Akt/HIF-1α Pathway.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Huang

    Full Text Available Though hypoxia has been implicated as a cause of inflammation, the underlying mechanism is not well understood. Folic acid has been shown to provide protection against oxidative stress and inflammation in patients with cardiovascular disease and various models approximating insult to tissue via inflammation. It has been reported that hypoxia-induced inflammation is associated with oxidative stress, upregulation of hypoxia-inducible factor 1-alpha (HIF-1α, and production of pro-inflammatory molecules. Whether folic acid protects human monocytic cells (THP-1 cells against hypoxia-induced damage, however, remains unknown. We used THP-1 cells to establish a hypoxia-induced cellular injury model. Pretreating THP-1 cells with folic acid attenuated hypoxia-induced inflammatory responses, including a decrease in protein and mRNA levels of interleukin (IL-1β and tumor necrosis factor-alpha (TNF-α, coupled with increased levels of IL-10. Folic acid also reduced hypoxia-induced Akt phosphorylation and decreased nuclear accumulation of HIF-1α protein. Both LY294002 (a selective inhibitor of phosphatidyl inositol-3 kinase, PI3K and KC7F2 (a HIF-1α inhibitor reduced levels of hypoxia-induced inflammatory cytokines. We also found that insulin (an Akt activator and dimethyloxallyl glycine (DMOG, a HIF-1α activator induced over-expression of inflammatory cytokines, which could be blocked by folic acid. Taken together, these findings demonstrate how folic acid attenuates the hypoxia-induced inflammatory responses of THP-1 cells through inhibition of the PI3K/Akt/HIF-1α pathway.

  11. Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers

    International Nuclear Information System (INIS)

    Bussink, Johan; Kaanders, Johannes H.A.M.; Kogel, Albert J. van der

    2003-01-01

    Background and purpose: Tumor oxygenation is recognized as an important determinant of the outcome of radiotherapy and possibly also of other treatment modalities in a number of tumor types and in particular in squamous cell carcinomas. The hypoxic status of various solid tumors has been related to a poor prognosis due to tumor progression towards a more malignant phenotype, with increased metastatic potential, and an increased resistance to treatment. It has been demonstrated in head and neck cancer that hypoxic radioresistance can be successfully counteracted by hypoxia modifying approaches. The microregional distribution and the level of tumor hypoxia depend on oxygen consumption and temporal and spatial variations in blood supply. It is unclear if severely hypoxic cells can resume clonogenicity when O 2 and nutrients become available again as a result of (treatment related) changes in the tumor microenvironment. Non-terminally differentiated hypoxic cells that are capable of proliferation are important for outcome because of their resistance to radiotherapy and possibly other cytotoxic treatments. Various exogenous and endogenous markers for hypoxia are currently available and can be studied in relation to each other, the tumor architecture and the tumor microenvironment. Use of nitroimidazole markers with immunohistochemical detection allows studying tumor cell hypoxia at the microscopic level. Co-registration with other microenvironmental parameters, such as vascular architecture (vascular density), blood perfusion, tumor cell proliferation and apoptosis, offers the possibility to obtain a comprehensive functional image of tumor patho-physiology and to study the effects of different modalities of cancer treatment. Conclusion: A number of functional microregional parameters have emerged that are good candidates for future use as indicators of tumor aggressiveness and treatment response. The key question is whether these parameters can be used as tools for

  12. Circulating levels of cell-derived microparticles are reduced by mild hypobaric hypoxia: data from a randomised controlled trial.

    Science.gov (United States)

    Ayers, Lisa; Stoewhas, Anne-Christin; Ferry, Berne; Latshang, Tsogyal D; Lo Cascio, Christian M; Sadler, Ross; Stadelmann, Katrin; Tesler, Noemi; Huber, Reto; Achermann, Peter; Bloch, Konrad E; Kohler, Malcolm

    2014-05-01

    Hypoxia is known to induce the release of microparticles in vitro. However, few publications have addressed the role of hypoxia in vivo on circulating levels of microparticles. This randomised, controlled, crossover trial aimed to determine the effect of mild hypoxia on in vivo levels of circulating microparticles in healthy individuals. Blood was obtained from 51 healthy male volunteers (mean age of 26.9 years) at baseline altitude (490 m) and after 24 and 48 h at moderate altitude (2,590 m). The order of altitude exposure was randomised. Flow cytometry was used to assess platelet-poor plasma for levels of circulating microparticles derived from platelets, endothelial cells, leucocytes, granulocytes, monocytes, red blood cells and procoagulant microparticles. Mean (standard deviation) oxygen saturation was significantly lower on the first and second day after arrival at 2,590 m, 91.0 (2.0) and 92.0 (2.0) %, respectively, compared to 490 m, 96 (1.0) %, p microparticles (annexin V+ -221/μl 95 % CI -370.8/-119.0, lactadherin+ -202/μl 95 % CI -372.2/-93.1), platelet-derived microparticles (-114/μl 95 % CI -189.9/-51.0) and red blood cell-derived microparticles (-81.4 μl 95 % CI -109.9/-57.7) after 48 h at moderate altitude was found. Microparticles derived from endothelial cells, granulocytes, monocytes and leucocytes were not significantly altered by exposure to moderate altitude. In healthy male individuals, mild hypobaric hypoxia, induced by a short-term stay at moderate altitude, is associated with lower levels of procoagulant microparticles, platelet-derived microparticles and red blood cell-derived microparticles, suggesting a reduction in thrombotic potential.

  13. δ-Tocotrienol Oxazine Derivative Antagonizes Mammary Tumor Cell Compensatory Response to CoCl2-Induced Hypoxia

    Directory of Open Access Journals (Sweden)

    Suryatheja Ananthula

    2014-01-01

    Full Text Available In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival. Studies were conducted to determine the role of δ-tocotrienol and a semisynthetic δ-tocotrienol oxazine derivative, compound 44, on +SA mammary tumor cell hypoxic response. Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response. CoCl2-induced hypoxia was also associated with a large increase in Akt/mTOR signaling, activation of downstream targets p70S6K and eIF-4E1, and a significant increase in VEGF production, and combined treatment with compound 44 blocked this response. Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6kinase and eIF-4E1. These findings demonstrate that treatment with the δ-tocotrienol oxazine derivative, compound 44, significantly attenuates +SA mammary tumor cell compensatory responses to hypoxia and suggests that this compound may provide benefit in the treatment of rapidly growing solid breast tumors.

  14. Silencing of NAC1 Expression Induces Cancer Cells Oxidative Stress in Hypoxia and Potentiates the Therapeutic Activity of Elesclomol

    Directory of Open Access Journals (Sweden)

    Yi-Jie Ren

    2017-11-01

    Full Text Available In order to survive under conditions of low oxygen, cancer cells can undergo a metabolic switch to glycolysis and suppress mitochondrial respiration in order to reduce oxygen consumption and prevent excessive amounts of reactive oxygen species (ROS production. Nucleus accumbens-1 (NAC1, a nuclear protein of the BTB/POZ gene family, has pivotal roles in cancer development. Here, we identified that NAC1-PDK3 axis as necessary for suppression of mitochondrial function, oxygen consumption, and more harmful ROS generation and protects cancer cells from apoptosis in hypoxia. We show that NAC1 mediates suppression of mitochondrial function in hypoxia through inducing expression of pyruvate dehydrogenase kinase 3 (PDK3 by HIF-1α at the transcriptional level, thereby inactivating pyruvate dehydrogenase and attenuating mitochondrial respiration. Re-expression of PDK3 in NAC1 absent cells rescued cells from hypoxia-induced metabolic stress and restored the activity of glycolysis in a xenograft mouse model, and demonstrated that silencing of NAC1 expression can enhance the antitumor efficacy of elesclomol, a pro-oxidative agent. Our findings reveal a novel mechanism by which NAC1 facilitates oxidative stress resistance during cancer progression, and chemo-resistance in cancer therapy.

  15. Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

    Science.gov (United States)

    Catalano, Rob D; Wilson, Martin R; Boddy, Sheila C; McKinlay, Andrew T M; Sales, Kurt J; Jabbour, Henry N

    2011-05-12

    The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

  16. Fluorescence Lifetime Imaging Microscopy (FLIM) as a Tool to Investigate Hypoxia-Induced Protein-Protein Interaction in Living Cells.

    Science.gov (United States)

    Schützhold, Vera; Fandrey, Joachim; Prost-Fingerle, Katrin

    2018-01-01

    Fluorescence resonance energy transfer (FRET) is widely used as a method to investigate protein-protein interactions in living cells. A FRET pair donor fluorophore in close proximity to an appropriate acceptor fluorophore transfers emission energy to the acceptor, resulting in a shorter lifetime of the donor fluorescence. When the respective FRET donor and acceptor are fused with two proteins of interest, a reduction in donor lifetime, as detected by fluorescence lifetime imaging microscopy (FLIM), can be taken as proof of close proximity between the fluorophores and therefore interaction between the proteins of interest. Here, we describe the usage of time-domain FLIM-FRET in hypoxia-related research when we record the interaction of the hypoxia-inducible factor-1 (HIF-1) subunits HIF-1α and HIF-1β in living cells in a temperature- and CO 2 -controlled environment under the microscope.

  17. Fatty Acid Uptake and Lipid Storage Induced by HIF-1α Contribute to Cell Growth and Survival after Hypoxia-Reoxygenation

    Directory of Open Access Journals (Sweden)

    Karim Bensaad

    2014-10-01

    Full Text Available Summary: An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3 and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1α (HIF-1α-dependent manner. There was a significant lipid droplet (LD accumulation in hypoxia that was time and O2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via β-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo. : Bensaad et al. now show that FABP3 and FABP7 are induced by HIF-1α and lead to a significant lipid droplet (LD accumulation in hypoxia. In hypoxia-reoxygenation, ATP production occurs via fatty acid β-oxidation or glycogen degradation in a cell-type-dependent manner, while inhibition of LD formation increases ROS toxicity and decreases cell survival in vitro and strongly impairs tumorigenesis in vivo.

  18. Multimodal hypoxia imaging and intensity modulated radiation therapy for unresectable non-small-cell lung cancer: the HIL trial

    Directory of Open Access Journals (Sweden)

    Askoxylakis Vasileios

    2012-09-01

    Full Text Available Abstract Background Radiotherapy, preferably combined with chemotherapy, is the treatment standard for locally advanced, unresectable non-small cell lung cancer (NSCLC. The tumor response to different therapy protocols is variable, with hypoxia known to be a major factor that negatively influences treatment effectiveness. Visualisation of tumor hypoxia prior to the use of modern radiation therapy strategies, such as intensity modulated radiation therapy (IMRT, might allow optimized dose applications to the target volume, leading to improvement of therapy outcome. 18 F-fluoromisonidazole dynamic positron emission tomography and computed tomography (18 F-FMISO dPET-CT and functional magnetic resonance imaging (functional MRI are attractive options for imaging tumor hypoxia. Methods/design The HIL trial is a single centre study combining multimodal hypoxia imaging with 18 F-FMISO dPET-CT and functional MRI, with intensity modulated radiation therapy (IMRT in patients with inoperable stage III NSCLC. 15 patients will be recruited in the study. All patients undergo initial FDG PET-CT and serial 18 F-FMISO dPET-CT and functional MRI before treatment, at week 5 of radiotherapy and 6 weeks post treatment. Radiation therapy is performed as inversely planned IMRT based on 4D-CT. Discussion Primary objectives of the trial are to characterize the correlation of 18 F-FMISO dPET-CT and functional MRI for tumor hypoxia imaging in NSCLC and evaluate possible effects of radiation therapy on tumor re-oxygenation. Further objectives include the generation of data regarding the prognostic value of 18 F-FMISO dPET-CT and functional MRI for locoregional control, progression free survival and overall survival of NSCLC treated with IMRT, which will form the basis for larger clinical trials focusing on possible interactions between tumor oxygenation and radiotherapy outcome. Trial registration The ClinicalTrials.gov protocol ID is NCT01617980

  19. Hypoxia-induced cell damage is reduced by mild hypothermia and postconditioning with catalase in-vitro: application of an enzyme based oxygen deficiency system.

    Science.gov (United States)

    Zitta, Karina; Meybohm, Patrick; Bein, Berthold; Rodde, Cornelia; Steinfath, Markus; Scholz, Jens; Albrecht, Martin

    2010-02-25

    Mild hypothermia and pharmacological postconditioning are widespread therapeutical treatment options that positively influence the clinical outcome after tissue hypoxia. In the study presented, a two-enzyme based in-vitro oxygen deficiency model in combination with cultured HT-1080 fibrosarcoma cells was employed to mimic the in-vivo situation of hypoxia and to evaluate the influence of mild hypothermia and postconditioning with catalase on hypoxia-mediated cell damage. Using the in-vitro oxygen deficiency model, partial pressure of oxygen was rapidly reduced to levels below 5mmHg in the culture media and cells responded with an increased expression of hypoxia inducible factor-1 on protein level. Hypoxia resulted in significant cell rounding and retraction of cytoplasmic cell extensions. Evaluation of cytotoxicity revealed a 3.5-fold increase in lactate dehydrogenase levels which was accompanied by 40-fold elevated levels of hydrogen peroxide. The hypoxia-induced increase of lactate dehydrogenase was 2.5-fold reduced in the hypothermia group, although morphological correlates of cytotoxicity were still visible. Hypothermia did not significantly influence hydrogen peroxide concentrations in the culture media. Pharmacological postconditioning with catalase however dose-dependently decreased hypoxia-induced lactate dehydrogenase release. This cytoprotective effect was accompanied by a dose-dependent, up to 50-fold reduction of hydrogen peroxide concentrations and retention of normal cell morphology. We suggest that the described in-vitro oxygen deficiency model is a convenient and simple culture system for the investigation of cellular and subcellular events associated with oxygen deficiency. Moreover, our in-vitro results imply that catalase postconditioning may be a promising approach to attenuate hypoxia-induced and hydrogen peroxide-mediated cell and tissue damage.

  20. Hypoxia-preconditioned mesenchymal stem cells ameliorate ischemia/reperfusion-induced lung injury.

    Directory of Open Access Journals (Sweden)

    Yung-Yang Liu

    Full Text Available Hypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs. However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels.I/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells and high (1×106 cells dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined.I/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS, pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism.Hypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis.

  1. Tumor microenvironment conditions alter Akt and Na+/H+ exchanger NHE1 expression in endothelial cells more than hypoxia alone

    DEFF Research Database (Denmark)

    Pedersen, Anna-Kathrine; Mendes Lopes de Melo, Joana; Mørup, Nina

    2017-01-01

    -ADP-ribose polymerase (PARP) cleavage consistent with inhibition of proliferation and induction of apoptosis. NHE1 knockdown, mimicking the effect of TME on NHE1 expression, reduced Ea.hy926 migration. TME effects on HIF-1α, VEGF, Akt, translation, proliferation or apoptosis markers were unaffected by NHE1 knockdown...... conditions. Here, we compared the effects of hypoxia and TME conditions on regulation of the Na+/H+ exchanger NHE1, Ser/Thr kinases Akt1–3, and downstream effectors in endothelial cells. Methods Human umbilical vein endothelial cells (HUVEC) and Ea.hy926 endothelial cells were exposed to simulated TME (1......% hypoxia, low serum, glucose, pH, high lactate) or 1% hypoxia for 24 or 48 h, with or without NHE1 inhibition or siRNA-mediated knockdown. mRNA and protein levels of NHE1, Akt1–3, and downstream effectors were assessed by qPCR and Western blotting, vascular endothelial growth factor (VEGF) release by ELISA...

  2. Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation

    Directory of Open Access Journals (Sweden)

    Alessandra Menon

    2018-01-01

    Full Text Available Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21% has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF, the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the “hypoxic niches” present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.

  3. Baicalin inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via the AKT/HIF-1α/p27-associated pathway.

    Science.gov (United States)

    Zhang, Lin; Pu, Zhichen; Wang, Junsong; Zhang, Zhifeng; Hu, Dongmei; Wang, Junjie

    2014-05-09

    Baicalin, a flavonoid compound purified from the dry roots of Scutellaria baicalensis Georgi, has been shown to possess various pharmacological actions. Previous studies have revealed that baicalin inhibits the growth of cancer cells through the induction of apoptosis. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by enhanced pulmonary artery smooth muscle cell (PASMCs) proliferation and suppressed apoptosis. However, the potential mechanism of baicalin in the regulation of PASMC proliferation and the prevention of cardiovascular diseases remains unexplored. To test the effects of baicalin on hypoxia, we used rats treated with or without baicalin (100 mg·kg⁻¹ each rat) at the beginning of the third week after hypoxia. Hemodynamic and pulmonary pathomorphology data showed that right ventricular systolic pressures (RVSP), the weight of the right ventricle/left ventricle plus septum (RV/LV + S) ratio and the medial width of pulmonary arterioles were much higher in chronic hypoxia. However, baicalin treatment repressed the elevation of RVSP, RV/LV + S and attenuated the pulmonary vascular structure remodeling (PVSR) of pulmonary arterioles induced by chronic hypoxia. Additionally, baicalin (10 and 20 μmol·L⁻¹) treatment suppressed the proliferation of PASMCs and attenuated the expression of hypoxia-inducible factor-α (HIF-α) under hypoxia exposure. Meanwhile, baicalin reversed the hypoxia-induced reduction of p27 and increased AKT/protein kinase B phosphorylation p-AKT both in vivo and in vitro. These results suggested that baicalin could effectively attenuate PVSR and hypoxic pulmonary hypertension.

  4. Propofol inhibits hypoxia/reoxygenation-induced human gastric epithelial cell injury by suppressing the Toll-like receptor 4 pathway

    Directory of Open Access Journals (Sweden)

    Jiao-Li Zhang

    2013-06-01

    Full Text Available This study aimed to investigate the role of the Toll-like receptor 4 (TLR4 pathway in normal human gastric epithelial (GES-1 cells under hypoxia/reoxygenation (H/R in vitro, and the effect of propofol on injured GES-1 cells as well as its possible mechanism. Before H/R induction, GES-1 cells were preconditioned with fat emulsion, propofol, or epigallocatechin gallate. Then cell viability, cell apoptosis, and related molecules in the cells were analyzed under experimental conditions. We found that propofol 50 μmol/L markedly inhibited the H/R injury under hypoxia 1.5 h/reoxygenation 2 hours by promoting GES-1 cell viability and decreasing cell apoptosis. The TLR4 signal may be involved in the protective effect of propofol against H/R injury. The malondialdehyde contents and superoxide dismutase activities were recovered under propofol preconditioning. In summary, propofol preconditioning may exert a protective effect on H/R injury in GES-1 cells and the mechanism may be via inhibition of the activated TLR4 signal under H/R conditions.

  5. The roles of transforming growth factor-β, Wnt, Notch and hypoxia on liver progenitor cells in primary liver tumours

    Science.gov (United States)

    BOGAERTS, ELIENE; HEINDRYCKX, FEMKE; VANDEWYNCKEL, YVES-PAUL; VAN GRUNSVEN, LEO A.; VAN VLIERBERGHE, HANS

    2014-01-01

    Primary liver tumours have a high incidence and mortality. The most important forms are hepatocellular carcinoma and intrahepatic cholangiocarcinoma, both can occur together in the mixed phenotype hepatocellular-cholangiocarcinoma. Liver progenitor cells (LPCs) are bipotential stem cells activated in case of severe liver damage and are capable of forming both cholangiocytes and hepatocytes. Possibly, alterations in Wnt, transforming growth factor-β, Notch and hypoxia pathways in these LPCs can cause them to give rise to cancer stem cells, capable of driving tumourigenesis. In this review, we summarize and discuss current knowledge on the role of these pathways in LPC activation and differentiation during hepatocarcinogenesis. PMID:24504124

  6. Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension

    Science.gov (United States)

    Chettimada, Sukrutha; Gupte, Rakhee; Rawat, Dhwajbahadur; Gebb, Sarah A.; McMurtry, Ivan F.

    2014-01-01

    Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which directs the cells to synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1α, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxia-induced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension. PMID:25480333

  7. Correlation of Hypoxia and Pro-senescence Protein Expression in Green Sea Turtle (Chelonia mydas Lung Epithelial and Dermal Fibroblast Cell Culture

    Directory of Open Access Journals (Sweden)

    Anggraini Barlian

    2018-03-01

    Full Text Available Recent studies have shown hypoxia-induced gene expression correlated with cellular senescence. HIF-1α (hypoxia-inducible factor 1-alpha, p53, and pRB were induced under hypoxia and correlated with cellular senescence. The localization and expression of HIF-1α, p53, and pRB in Chelonia mydas lung epithelial and dermal fibroblast cell cultures were analyzed under normoxic and hypoxic conditions (at 4 and 24 hours. Human dermal fibroblast was used for comparison purposes. Protein localization was analyzed with immunocytochemistry, while protein expression was analyzed with the Western blot and enhanced chemiluminescence (ECL method. HIF-1α, p53, and pRB were localized in the nuclei of the C. mydas cell cultures treated with hypoxia. The C. mydas lung epithelial cell cultures had a higher increase of HIF-1α expression than the human dermal fibroblast cell culture. The hypoxic conditions did not affect p53 expression significantly in C. mydas lung epithelial and dermal fibroblast cell cultures. Meanwhile, pRB expression changed significantly under hypoxia in the C. mydas dermal fibroblast cells. Expression of p53 and pRB in the human cell cultures was higher than in the C. mydas cell cultures. This research suggests that C. mydas and human cell cultures have different pro-senescence protein expression responses under hypoxic conditions.

  8. Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats

    DEFF Research Database (Denmark)

    Jensen, P O; Mortensen, B T; Hodgkiss, R J

    2000-01-01

    for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest......-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27...... in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From...

  9. Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Zengqi Tan

    2018-03-01

    Full Text Available The epithelial-mesenchymal transition (EMT process plays a key role in many biological processes, including tissue fibrosis, metastatic diseases, and cancer progression. EMT can be induced by certain factors, notably hypoxia, in the tumor microenvironment. Aberrant levels of certain N-glycans is associated with cancer progression. We used an integrated strategy (mass spectrometry in combination with lectin microarray analysis to elucidate aberrant glycosylation in a hypoxia-induced EMT model using breast cancer cell lines MCF7 and MDA-MB-231. The model showed reduced levels of bisecting GlcNAc structures, and downregulated expression of the corresponding glycosyltransferase MGAT3. MGAT3 overexpression in MCF7 suppressed cell migration, proliferation, colony formation, expression of EMT markers, and AKT signaling pathway, whereas MGAT3 knockdown (shRNA silencing had opposite effects. Our findings clearly demonstrate the functional role (and effects of dysregulation of bisecting GlcNAc structures in hypoxia-induced EMT, and provide a useful basis for further detailed studies of physiological functions of these structures in breast cancer.

  10. Delta-9-tetrahydrocannabinol protects cardiac cells from hypoxia via CB2 receptor activation and nitric oxide production.

    Science.gov (United States)

    Shmist, Yelena A; Goncharov, Igor; Eichler, Maor; Shneyvays, Vladimir; Isaac, Ahuva; Vogel, Zvi; Shainberg, Asher

    2006-02-01

    Delta-9-tetrahydrocannabinol (THC), the major active component of marijuana, has a beneficial effect on the cardiovascular system during stress conditions, but the defence mechanism is still unclear. The present study was designed to investigate the central (CB1) and the peripheral (CB2) cannabinoid receptor expression in neonatal cardiomyoctes and possible function in the cardioprotection of THC from hypoxia. Pre-treatment of cardiomyocytes that were grown in vitro with 0.1 - 10 microM THC for 24 h prevented hypoxia-induced lactate dehydrogenase (LDH) leakage and preserved the morphological distribution of alpha-sarcomeric actin. The antagonist for the CB2 (10 microM), but not CB1 receptor antagonist (10 microM) abolished the protective effect of THC. In agreement with these results using RT-PCR, it was shown that neonatal cardiac cells express CB2, but not CB1 receptors. Involvement of NO in the signal transduction pathway activated by THC through CB2 was examined. It was found that THC induces nitric oxide (NO) production by induction of NO synthase (iNOS) via CB2 receptors. L-NAME (NOS inhibitor, 100 microM) prevented the cardioprotection provided by THC. Taken together, our findings suggest that THC protects cardiac cells against hypoxia via CB2 receptor activation by induction of NO production. An NO mechanism occurs also in the classical pre-conditioning process; therefore, THC probably pre-trains the cardiomyocytes to hypoxic conditions.

  11. The Neuroprotective Potential of Rho-Kinase Inhibition in Promoting Cell Survival and Reducing Reactive Gliosis in Response to Hypoxia in Isolated Bovine Retina

    Directory of Open Access Journals (Sweden)

    Aizhan Alt

    2013-07-01

    Full Text Available Aims: To investigate the outcomes of Rho-kinase inhibition in the electrophysiological ex vivo model of the isolated perfused vertebrate retina under hypoxia. Methods: Bovine retinas were perfused with an oxygen saturated nutrient solution with or without the Rho-kinase inhibitor H-1152P. The retinas were stimulated repeatedly until stable amplitudes were reached and the electroretinogram was recorded at five minute intervals. Hypoxia was induced for 15, 30, and 45 minutes, after which the oxygen saturation was restored. The extent of the cell damage and glial reactivity was determined by Ethidium homodimer-1 staining, immunohistochemistry, and Western blot. Results: Hypoxia caused a time-dependent reduction of the b-wave amplitudes, which could not be prevented by the H-1152P. Although the Rho-kinase inhibitor maintained higher b-wave amplitudes, these effects did not reach statistical significance. Hypoxia also resulted in an increase in cell damage and the activation of the glial cells in the untreated retinas whereas the administration of H-1152P significantly reduced the extent of these events. Conclusion: H-1152P exerted a neuroprotective effect against necrosis on the isolated bovine retina under hypoxia together with a reduction in glial cell reactivity. However, the inhibitor could not prevent the hypoxia induced retinal dysfunction possibly due to the interference with synaptic modulation.

  12. [Effects of hypoxia inducible factor-1α on P311 and its influence on the migration of murine epidermal stem cells].

    Science.gov (United States)

    Xu, Z D; Li, H S; Wang, S; He, W F; Wu, J; Luo, G X

    2017-05-20

    Objective: To explore the effects of hypoxia inducible factor-1α (HIF-1α) on P311 and its influence on the migration of murine epidermal stem cells (ESCs) under hypoxia in vitro. Methods: Two kinds of murine ESCs were isolated and obtained from 15 neonatal wild-type C57BL/6J mice and 5 congeneric source P311 gene knock-out mice, respectively. The first passage of cells were used in the following experiments after morphologic observation and detection of expression of cell surface markers CD71 and CD49f with flow cytometer. (1) After cell scratch assay, according to the random number table (the same dividing method below), ESCs of P311 gene knock-out mice were divided into normoxia group (cells were cultured with complete medium in normoxic carbon dioxide incubator, and the subsequent normoxic treatments were the same) and hypoxia group (cells were cultured in hypoxic carbon dioxide incubator containing 1% oxygen, and the subsequent hypoxic treatments were the same), with 12 inserts in each group. ESCs of wild-type mice were divided into normoxia group, pure hypoxia group, dimethyl sulfoxide (DMSO) control group (2 μL DMSO solvent was added for 1 h of normoxia treatment before hypoxia treatment), HIF-1α inhibitor group (cells were treated with 11 μmol/L HIF-1 inhibitor of 2 μL under normoxia condition for 1 h before hypoxia treatment), HIF-1α stabilizer group (the cells were treated with 2 μmol/L FG-4592 of 2 μL under normoxia condition for 1 h before hypoxia treatment), with 12 inserts in each group. Three inserts of each time point in each group were adopted respectively to measure the residual width of scratch under inverted phase contrast microscope at post scratch hour (PSH) 0 (immediately), 12, 24, and 48. (2) After hypoxia treatment, the protein level of HIF-1α in ESCs of wild-type mice was detected by Western blotting at post hypoxia hour (PHH) 0, 12, 24, and 48. (3) ESCs of wild-type mice were divided into pure hypoxia group, DMSO control group

  13. ERK1/2 and Akt phosphorylation were essential for MGF E peptide regulating cell morphology and mobility but not proangiogenic capacity of BMSCs under severe hypoxia.

    Science.gov (United States)

    Sha, Yongqiang; Yang, Li; Lv, Yonggang

    2018-02-13

    Severe hypoxia inhibits the adhesion and mobility of bone marrow-derived mesenchymal stem cells (BMSCs) and limits their application in bone tissue engineering. In this study, CoCl 2 was used to simulate severe hypoxia and the effects of mechano-growth factor (MGF) E peptide on the morphology, adhesion, migration, and proangiogenic capacity of BMSCs under hypoxia were measured. It was demonstrated that severe hypoxia (500-μM CoCl 2 ) significantly caused cell contraction and reduced cell area, roundness, adhesion, and migration of BMSCs. RhoA and ROCK1 expression levels were upregulated by severe hypoxia, but p-RhoA and mobility-relevant protein (integrin β1, p-FAK and fibronectin) expression levels in BMSCs were inhibited. Fortunately, MGF E peptide could restore all abovementioned indexes except RhoA expression. MEK-ERK1/2 pathway was involved in MGF E peptide regulating cell morphological changes, mobility, and relevant proteins (except p-FAK). PI3K-Akt pathway was involved in MGF E peptide regulating cell area, mobility, and relevant proteins. Besides, severe hypoxia upregulated vascular endothelial growth factor α expression but was harmful for proangiogenic capacity of BMSCs. Our study suggested that MGF E peptide might be helpful for the clinical application of tissue engineering strategy in bone defect repair. Sever hypoxia impairs bone defect repair with bone marrow-derived mesenchymal stem cells (BMSCs). This study proved that mechano-growth factor E (MGF E) peptide could improve the severe hypoxia-induced cell contraction and decline of cell adhesion and migration of BMSCs. Besides, MGF E peptide weakened the effects of severe hypoxia on the cytoskeleton arrangement- and mobility-relevant protein expression levels in BMSCs. The underlying molecular mechanism was also verified. Finally, it was confirmed that MGF E peptide showed an adverse effect on the expression level of vascular endothelial growth factor α in BMSCs under severe hypoxia but could

  14. Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Can [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Zhang, Li-Yang [Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Cancer Research Institute, Central South University, 110 Xiang Ya Road, Changsha 410078 (China); Chen, Hong [Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Xiao, Ling [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Liu, Xian-Peng, E-mail: xliu@lsuhsc.edu [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (United States); Zhang, Jian-Xiang, E-mail: jianxiangzhang@yahoo.cn [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.

  15. Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells.

    Science.gov (United States)

    Armitage, Emily G; Kotze, Helen L; Allwood, J William; Dunn, Warwick B; Goodacre, Royston; Williams, Kaye J

    2015-10-28

    Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments.

  16. Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

    Science.gov (United States)

    Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

    2017-10-01

    Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S

  17. Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

    Science.gov (United States)

    Correia, Cláudia; Serra, Margarida; Espinha, Nuno; Sousa, Marcos; Brito, Catarina; Burkert, Karsten; Zheng, Yunjie; Hescheler, Jürgen; Carrondo, Manuel J T; Sarić, Tomo; Alves, Paula M

    2014-12-01

    Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

  18. Oroxylin A regulates glucose metabolism in response to hypoxic stress with the involvement of Hypoxia-inducible factor-1 in human hepatoma HepG2 cells.

    Science.gov (United States)

    Dai, Qinsheng; Yin, Qian; Wei, Libin; Zhou, Yuxin; Qiao, Chen; Guo, Yongjian; Wang, Xiaotang; Ma, Shiping; Lu, Na

    2016-08-01

    Metabolic alteration in cancer cells is one of the most conspicuous characteristics that distinguish cancer cells from normal cells. In this study, we investigated the influence and signaling ways of oroxylin A affecting cancer cell energy metabolism under hypoxia. The data showed that oroxylin A remarkably reduced the generation of lactate and glucose uptake under hypoxia in HepG2 cells. Moreover, oroxylin A inhibited HIF-1α expression and its stability. The downstream targets (PDK1, LDHA, and HK II), as well as their mRNA levels were also suppressed by oroxylin A under hypoxia. The silencing or the overexpression of HIF-1α assays suggested that HIF-1α is required for metabolic effect of oroxylin A in HepG2 cells during hypoxia. Furthermore, oroxylin A could reduce the expression of complex III in mitochondrial respiratory chain, and then decrease the accumulation of ROS at moderate concentrations (0-50 µM) under hypoxia, which was benefit for its inhibition on glycolytic activity by decreasing ROS-mediated HIF-1 expression. Besides, oroxylin A didn't cause the loss of MMP under hypoxia and had no obvious effects on the expression of OXPHOS complexes, suggesting that oroxylin A did not affect mitochondrial mass at the moderate stress of oroxylin A. The suppressive effect of oroxylin A on glycolysis led to a significantly repress of ATP generation, for ATP generation mostly depends on glycolysis in HepG2 cells. This study revealed a new aspect of glucose metabolism regulation of oroxylin A under hypoxia, which may contribute to its new anticancer mechanism. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. 17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

    Science.gov (United States)

    Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

    2010-03-01

    The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

  20. Survival of cancer stem cells under hypoxia and serum depletion via decrease in PP2A activity and activation of p38-MAPKAPK2-Hsp27.

    Directory of Open Access Journals (Sweden)

    Shih-Pei Lin

    Full Text Available Hypoxia and serum depletion are common features of solid tumors that occur upon antiangiogenesis, irradiation and chemotherapy across a wide variety of malignancies. Here we show that tumor cells expressing CD133, a marker for colorectal cancer initiating or stem cells, are enriched and survive under hypoxia and serum depletion conditions, whereas CD133- cells undergo apoptosis. CD133+ tumor cells increase cancer stem cell and epithelial-mesenchymal transition properties. Moreover, via screening a panel of tyrosine and serine/threonine kinase pathways, we identified Hsp27 is constitutively activated in CD133+ cells rather than CD133- cell under hypoxia and serum depletion conditions. However, there was no difference in Hsp27 activation between CD133+ and CD133- cells under normal growth condition. Hsp27 activation, which was mediated by the p38MAPK-MAPKAPK2-Hsp27 pathway, is required for CD133+ cells to inhibit caspase 9 and 3 cleavage. In addition, inhibition of Hsp27 signaling sensitizes CD133+ cells to hypoxia and serum depletion -induced apoptosis. Moreover, the antiapoptotic pathway is also activated in spheroid culture-enriched CD133+ cancer stem cells from a variety of solid tumor cells including lung, brain and oral cancer, suggesting it is a common pathway activated in cancer stem cells from multiple tumor types. Thus, activation of PP2A or inactivation of the p38MAPK-MAPKAPK2-Hsp27 pathway may develop new strategies for cancer therapy by suppression of their TIC population.

  1. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

    International Nuclear Information System (INIS)

    Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira; Shigeno, Keiji; Nakamura, Tatsuo

    2012-01-01

    Highlights: ► Very rapid generation of human iPS cells under optimized conditions. ► Five chemical inhibitors under hypoxia boosted reprogramming. ► We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of i

  2. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Hidenori [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Hashimoto, Yoshiya [Department of Biomaterials, Osaka Dental University, 8-1, Hanazonocho, Kuzuha, Hirakatashi, Osaka 573-1121 (Japan); Nakada, Akira; Shigeno, Keiji [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Nakamura, Tatsuo, E-mail: nakamura@frontier.kyoto-u.ac.jp [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly

  3. Suppression of ERK1/2 and hypoxia pathways by four Phyllanthus species inhibits metastasis of human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Sau H. Lee

    2016-10-01

    Full Text Available Chemotherapies remain far from ideal due to drug resistance; therefore, novel chemotherapeutic agents with higher effectiveness are crucial. The extracts of four Phyllanthus species, namely Phyllanthus niruri, Phyllanthus urinaria, Phyllanthus watsonii, and Phyllanthus amarus, were shown to induce apoptosis and inhibit metastasis of breast carcinoma cells (MCF-7. The main objective of this study was to determine the pathways utilized by these four Phyllanthus species to exert anti-metastatic activities. A cancer 10-pathway reporter was used to investigate the pathways affected by the four Phyllanthus species. Results indicated that these Phyllanthus species suppressed breast carcinoma metastasis and proliferation by suppressing matrix metalloprotein 2 and 9 expression via inhibition of the extracellular signal-related kinase (ERK pathway. Additionally, inhibition of hypoxia-inducible factor 1-α in the hypoxia pathway caused reduced vascular endothelial growth factor and inducible nitric oxide synthase expression, resulting in anti-angiogenic effects and eventually anti-metastasis. Two-dimensional gel electrophoresis identified numerous proteins suppressed by these Phyllanthus species, including invasion proteins, anti-apoptotic protein, protein-synthesis proteins, angiogenic and mobility proteins, and various glycolytic enzymes. Our results indicated that ERK and hypoxia pathways are the most likely targets of the four Phyllanthus species for the inhibition of MCF-7 metastasis.

  4. Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

    Science.gov (United States)

    Tsuzuki, Tomoko; Okada, Hidetaka; Cho, Hisayuu; Tsuji, Shoko; Nishigaki, Akemi; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2012-02-01

    Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl₂; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl₂ in a dose-dependent manner. CoCl₂ significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P hypoxic conditions that could influence angiogenesis in the human endometrium.

  5. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis

    NARCIS (Netherlands)

    Greijer, A.E.; Wall, E. van der

    2004-01-01

    Apoptosis can be induced in response to hypoxia. The severity of hypoxia determines whether cells become apoptotic or adapt to hypoxia and survive. A hypoxic environment devoid of nutrients prevents the cell undergoing energy dependent apoptosis and cells become necrotic. Apoptosis regulatory

  6. TGF-β and Hypoxia/Reoxygenation Promote Radioresistance of A549 Lung Cancer Cells through Activation of Nrf2 and EGFR

    Directory of Open Access Journals (Sweden)

    Sae-lo-oom Lee

    2016-01-01

    Full Text Available Although many studies have examined the roles of hypoxia and transforming growth factor- (TGF- β separately in the tumor microenvironment, the effects of simultaneous treatment with hypoxia/reoxygenation and TGF-β on tumor malignancy are unclear. Here, we investigated the effects of redox signaling and oncogenes on cell proliferation and radioresistance in A549 human lung cancer cells in the presence of TGF-β under hypoxia/reoxygenation conditions. Combined treatment with TGF-β and hypoxia activated epidermal growth factor receptor (EGFR and nuclear factor (erythroid-derived 2-like 2 (Nrf2, a redox-sensitive transcription factor. Interestingly, Nrf2 knockdown suppressed the effects of combined treatment on EGFR phosphorylation. In addition, blockade of EGFR signaling also suppressed induction of Nrf2 following combined treatment with hypoxia and TGF-β, indicating that the combined treatment induced positive crosstalk between Nrf2 and EGFR. TGF-β and hypoxia/reoxygenation increased the accumulation of reactive oxygen species (ROS, while treatment with N-acetyl-L-cysteine abolished the activation of Nrf2 and EGFR. Treatment with TGF-β under hypoxic conditions increased the proliferation of A549 cells compared with that after vehicle treatment. Moreover, cells treated with the combined treatment exhibited resistance to ionizing radiation (IR, and knockdown of Nrf2 increased IR-induced cell death under these conditions. Thus, taken together, our findings suggested that TGF-β and hypoxia/reoxygenation promoted tumor progression and radioresistance of A549 cells through ROS-mediated activation of Nrf2 and EGFR.

  7. LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

    Science.gov (United States)

    Sato, Mariko; Hirose, Katsumi; Kashiwakura, Ikuo; Aoki, Masahiko; Kawaguchi, Hideo; Hatayama, Yoshiomi; Akimoto, Hiroyoshi; Narita, Yuichiro; Takai, Yoshihiro

    2015-09-01

    Hypoxia‑inducible factor 1 (HIF‑1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF‑1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF‑1α. A clear anti‑tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF‑1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF‑1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel‑Lindau protein. In addition, at this concentration, LW6 induced hypoxia‑selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6‑treated hypoxic A549 cells and LW6 induced a hypoxia‑selective increase of mitochondrial O2•‑. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.

  8. MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xue-Feng [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Respiration, Qinghai Provincial People' s Hospital, Xining (China); Wang, Hua; Xiao, Feng-Jun [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Yin, Yue [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Hematology, Peking University First Hospital, Beijing (China); Xu, Qin-Qin [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Ge, Ri-Li, E-mail: geriligao@hotmail.com [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Wang, Li-Sheng, E-mail: wangls@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2016-02-12

    MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases. - Highlights: • miR-486 is a hypoxia-induced miRNA. • miR-486 regulates the secretion of HGF and VEGF, promotes proliferation, and inhibits apoptosis of BM-MSCs. • miR-486 enhances PI3K/AKT activity signaling by targeting PTEN molecule.

  9. MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal

    International Nuclear Information System (INIS)

    Shi, Xue-Feng; Wang, Hua; Xiao, Feng-Jun; Yin, Yue; Xu, Qin-Qin; Ge, Ri-Li; Wang, Li-Sheng

    2016-01-01

    MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases. - Highlights: • miR-486 is a hypoxia-induced miRNA. • miR-486 regulates the secretion of HGF and VEGF, promotes proliferation, and inhibits apoptosis of BM-MSCs. • miR-486 enhances PI3K/AKT activity signaling by targeting PTEN molecule.

  10. Tanshinone IIA inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via Akt/Skp2/p27-associated pathway.

    Directory of Open Access Journals (Sweden)

    Ying Luo

    Full Text Available We previously showed that tanshinone IIA ameliorated the hypoxia-induced pulmonary hypertension (HPH partially by attenuating pulmonary artery remodeling. The hypoxia-induced proliferation of pulmonary artery smooth muscle cells (PASMCs is one of the major causes for pulmonary arterial remodeling, therefore the present study was performed to explore the effects and underlying mechanism of tanshinone IIA on the hypoxia-induced PASMCs proliferation. PASMCs were isolated from male Sprague-Dawley rats and cultured in normoxic (21% or hypoxic (3% condition. Cell proliferation was measured with 3 - (4, 5 - dimethylthiazal - 2 - yl - 2, 5 - diphenyltetrazoliumbromide assay and cell counting. Cell cycle was measured with flow cytometry. The expression of of p27, Skp-2 and the phosphorylation of Akt were measured using western blot and/or RT-PCR respectively. The results showed that tanshinone IIA significantly inhibited the hypoxia-induced PASMCs proliferation in a concentration-dependent manner and arrested the cells in G1/G0-phase. Tanshinone IIA reversed the hypoxia-induced reduction of p27 protein, a cyclin-dependent kinase inhibitor, in PASMCs by slowing down its degradation. Knockdown of p27 with specific siRNA abolished the anti-proliferation of tanshinone IIA. Moreover, tanshinone IIA inhibited the hypoxia-induced increase of S-phase kinase-associated protein 2 (Skp2 and the phosphorylation of Akt, both of which are involved in the degradation of p27 protein. In vivo tanshinone IIA significantly upregulated the hypoxia-induced p27 protein reduction and downregulated the hypoxia-induced Skp2 increase in pulmonary arteries in HPH rats. Therefore, we propose that the inhibition of tanshinone IIA on hypoxia-induce PASMCs proliferation may be due to arresting the cells in G1/G0-phase by slowing down the hypoxia-induced degradation of p27 via Akt/Skp2-associated pathway. The novel information partially explained the anti-remodeling property of

  11. Cerebral Hypoxia

    Science.gov (United States)

    ... off. When hypoxia lasts for longer periods of time, it can cause coma, seizures, and even brain death. In brain death, there is no measurable activity in the brain, although cardiovascular function is preserved. Life support is required for respiration. × Definition Cerebral hypoxia ...

  12. Radiosensitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide under oxia and hypoxia in human colon cancer cells

    International Nuclear Information System (INIS)

    Itani, Wafica; Geara, Fady; Haykal, Joelle; Haddadin, Makhluf; Gali-Muhtasib, Hala

    2007-01-01

    The sensitizing effects of 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ) and ionizing radiation (IR) were determined in four colon cancer cells and in FHs74Int normal intestinal cells. Cell cycle modulation, TUNEL assay, clonogenic survival and DNA damage were examined under oxia or hypoxia. Effects on apoptotic molecules and on p-Akt and Cox-2 protein expression were investigated. The four cell lines responded differently to DCQ+IR; HT-29 cells were most resistant. Combination treatment caused significant increases in preG 1 (apoptosis) in HCT-116, while G 2 /M arrest occurred in DLD-1. DCQ potentiated IR effects more so under hypoxia than oxia. Pre-exposure of DLD-1 to hypoxia induced 30% apoptosis, and G 2 /M arrest in oxia. The survival rate was 50% lower in DCQ+IR than DCQ alone and this rate further decreased under hypoxia. FHs74Int normal intestinal cells were more resistant to DCQ+IR than cancer cells.Greater ssDNA damage occurred in DLD-1 exposed to DCQ+IR under hypoxia than oxia. In oxia, p-Akt protein expression increased upon IR exposure and drug pre-treatment inhibited this increase. In contrast, in hypoxia, exposure to IR reduced p-Akt protein and DCQ restored its expression to the untreated control. Apoptosis induced in hypoxic DLD-1 cells was independent of p53-p21 modulation but was associated with an increase in Bax/Bcl-2 ratio and the inhibition of the Cox-2 protein. DCQ is a hypoxic cell radiosensitizer in DLD-1 human colon cancer cells

  13. Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

    Directory of Open Access Journals (Sweden)

    Reint K Jellema

    Full Text Available Hypoxic-ischemic encephalopathy (HIE in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6 MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI, in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

  14. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  15. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    International Nuclear Information System (INIS)

    Luo, Chunxia; Yi, Bin; Bai, Li; Xia, Yongzhi; Wang, Guansong; Qian, Guisheng; Feng, Hua

    2010-01-01

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  16. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-01-01

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β 1 (TGF-β 1 )-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β 1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β 1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β 1

  17. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  18. STARS knockout attenuates hypoxia-induced pulmonary arterial hypertension by suppressing pulmonary arterial smooth muscle cell proliferation.

    Science.gov (United States)

    Shi, Zhaoling; Wu, Huajie; Luo, Jianfeng; Sun, Xin

    2017-03-01

    STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein, which expressed early in cardiac development and involved in pathological remodeling. Abundant evidence indicated that STARS could regulate cell proliferation, but it's exact function remains unclear. In this study, we aimed to investigate the role of STARS in the proliferation of pulmonary arterial smooth muscle cells (PASMC) and the potential effect on the progression of pulmonary arterial hypertension (PAH). In this study, we established a PAH mouse model through chronic hypoxia exposure as reflected by the increased RVSP and RVHI. Western blot and RT-qPCR detected the increased STARS protein and mRNA levels in PAH mice. Next, we cultured the primary PASMC from PAH mice. After STARS overexpression in PASMC, STARS, SRF and Egr-1 were up-regulated significantly. The MTT assay revealed an increase in cell proliferation. Flow cytometry showed a marked inhibition of cell apoptosis. However, STARS silence in PASMC exerted opposite effects with STARS overexpression. SRF siRNA transfection blocked the effects of STARS overexpression in PASMC. In order to further confirm the role of STARS in PAH mice in vivo, we exposed STARS knockout mice to hypoxia and found lower RVSP and RVHI in knockout mice as compared with controls. Our results not only suggest that STARS plays a crucial role in the development of PAH by increasing the proliferation of PASMC through activation of the SRF/Egr-1 pathway, but also provides a new mechanism for hypoxia-induced PAH. In addition, STARS may represent a potential treatment target. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  19. Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

    Directory of Open Access Journals (Sweden)

    Rob D Catalano

    Full Text Available The prostaglandin endoperoxide synthase (PTGS pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG E(2. PTGS2 expression and PGE(2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2 regulate endometrial tumour growth is unknown. Here we investigated (a the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3 and PGE receptors (PTGER1-4 in endometrial adenocarcinomas compared with normal endometrium and (b the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

  20. Hypoxia-elicited impairment of cell wall integrity, glycosylation precursor synthesis, and growth in scaled-up high-cell density fed-batch cultures of Saccharomyces cerevisiae.

    Science.gov (United States)

    Aon, Juan C; Sun, Jianxin; Leighton, Julie M; Appelbaum, Edward R

    2016-08-15

    In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW. We tested the cell wall integrity by probing the cell wall sensitivity to Zymolyase. Also exometabolomics data showed changes in supply of precursors for the glycosylation pathway. The results show a more sensitive cell wall later in the production process at industrial scale, while the sensitivity at early time points was similar at both scales. We also report exometabolomics data, in particular a link with the protein glycosylation pathway. Significantly lower levels of Man6P and progressively higher GDP-mannose indicated partially impaired incorporation of this sugar nucleotide during co- or post-translational protein glycosylation pathways at the 10,000 L compared to the 10 L scale. This impairment in glycosylation would be expected to affect cell wall integrity. Although cell viability from samples obtained at both scales were similar, cells harvested from 10 L bioreactors were able to re-initiate growth faster in fresh shake flask media than those harvested from the industrial scale. The results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.

  1. Hypoxia Upregulates Mitotic Cyclins Which Contribute to the Multipotency of Human Mesenchymal Stem Cells by Expanding Proliferation Lifespan.

    Science.gov (United States)

    Lee, Janet; Kim, Hyun-Soo; Kim, Su-Min; Kim, Dong-Ik; Lee, Chang-Woo

    2018-02-21

    Hypoxic culture is widely recognized as a method to efficiently expand human mesenchymal stem cells (MSCs) without loss of stem cell properties. However, the molecular basis of how hypoxia priming benefits MSC expansion remains unclear. In this report, our systemic quantitative proteomic and RT-PCR analyses revealed the involvement of hypoxic conditioning activated genes in the signaling process of the mitotic cell cycle. Introduction of screened two mitotic cyclins, CCNA2 and CCNB1, significantly extended the proliferation lifespan of MSCs in normoxic condition. Our results provide important molecular evidence that multipotency of human MSCs by hypoxic conditioning is determined by the mitotic cell cycle duration. Thus, the activation of mitotic cyclins could be a potential strategy to the application of stem cell therapy.

  2. Lutein Attenuates Both Apoptosis and Autophagy upon Cobalt (II) Chloride-Induced Hypoxia in Rat Műller Cells.

    Science.gov (United States)

    Fung, Frederic K C; Law, Betty Y K; Lo, Amy C Y

    2016-01-01

    Retinal ischemia/reperfusion injury is a common feature of various retinal diseases such as glaucoma and diabetic retinopathy. Lutein, a potent anti-oxidant, is used to improve visual function in patients with age-related macular degeneration (AMD). Lutein attenuates apoptosis, oxidative stress and inflammation in animal models of acute retinal ischemia/hypoxia. Here, we further show that lutein improved Műller cell viability and enhanced cell survival upon hypoxia-induced cell death through regulation of intrinsic apoptotic pathway. Moreover, autophagy was activated upon treatment of cobalt (II) chloride, indicating that hypoxic injury not only triggered apoptosis but also autophagy in our in vitro model. Most importantly, we report for the first time that lutein treatment suppressed autophagosome formation after hypoxic insult and lutein administration could inhibit autophagic event after activation of autophagy by a pharmacological approach (rapamycin). Taken together, lutein may have a beneficial role in enhancing glial cell survival after hypoxic injury through regulating both apoptosis and autophagy.

  3. Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-β/Smad pathway activation.

    Science.gov (United States)

    Nakagawa, Yoko; Ashihara, Eishi; Yao, Hisayuki; Yokota, Asumi; Toda, Yuki; Miura, Yasuo; Nakata, Susumu; Hirai, Hideyo; Maekawa, Taira

    2018-02-05

    The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O 2 ). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O 2 ). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-β/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-β/Smad pathway activation. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

    Science.gov (United States)

    Deep, Gagan; Kumar, Rahul; Nambiar, Dhanya K; Jain, Anil K; Ramteke, Anand M; Serkova, Natalie J; Agarwal, Chapla; Agarwal, Rajesh

    2017-03-01

    Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O 2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Influence of in vivo hypobaric hypoxia on function of lymphocytes, neutrocytes, natural killer cells, and cytokines

    DEFF Research Database (Denmark)

    Klokker, M; Kharazmi, A; Galbo, H

    1993-01-01

    We have investigated the effects of short-term hypoxia in vivo on the human cellular immune system. Seven young healthy volunteers were placed in a decompression chamber (380 Torr) for 20 min with or without supplemental O2. The leukocyte concentration increased during hypobaric conditions becaus...

  6. Frequency and magnitude of intermittent hypoxia modulate endothelial wound healing in a cell culture model of sleep apnea.

    Science.gov (United States)

    Campillo, Noelia; Falcones, Bryan; Montserrat, Josep M; Gozal, David; Obeso, Ana; Gallego-Martin, Teresa; Navajas, Daniel; Almendros, Isaac; Farré, Ramon

    2017-11-01

    Intermittent hypoxia (IH) has been implicated in the cardiovascular consequences of obstructive sleep apnea (OSA). However, the lack of suitable experimental systems has precluded assessment as to whether IH is detrimental, protective, or both for the endothelium. The aim of the work was to determine the effects of frequency and amplitude of IH oxygenation swings on aortic endothelial wound healing. Monolayers of human primary endothelial cells were wounded and subjected to constant oxygenation (1%, 4%, 13%, or 20% O 2 ) or IH at different frequencies (0.6, 6, or 60 cycles/h) and magnitude ranges (13-4% O 2 or 20-1% O 2 ), using a novel well-controlled system, with wound healing being measured after 24 h. Cell monolayer repair was similar at 20% O 2 and 13% O 2 , but was considerably increased (approximately twofold) in constant hypoxia at 4% O 2 The magnitude and frequency of IH considerably modulated wound healing. Cycles ranging 13-4% O 2 at the lowest frequency (0.6 cycles/h) accelerated endothelial wound healing by 102%. However, for IH exposures consisting of 20% to 1% O 2 oscillations, wound closure was reduced compared with oscillation in the 13-4% range (by 74% and 44% at 6 cycles/h and 0.6 cycles/h, respectively). High-frequency IH patterns simulating severe OSA (60 cycles/h) did not significantly modify endothelial wound closure, regardless of the oxygenation cycle amplitude. In conclusion, the frequency and magnitude of hypoxia cycling in IH markedly alter wound healing responses and emerge as key factors determining how cells will respond in OSA. NEW & NOTEWORTHY Intermittent hypoxia (IH) induces cardiovascular consequences in obstructive sleep apnea (OSA) patients. However, the vast array of frequencies and severities of IH previously employed in OSA-related experimental studies has led to controversial results on the effects of IH. By employing an optimized IH experimental system here, we provide evidence that the frequency and magnitude of IH

  7. Ginsenoside Rb1 inhibits hypoxia-induced epithelial-mesenchymal transition in ovarian cancer cells by regulating microRNA-25.

    Science.gov (United States)

    Liu, Dan; Liu, Ting; Teng, Yue; Chen, Wei; Zhao, Le; Li, Xu

    2017-10-01

    Metastasis frequently occurs in advanced ovarian cancer, which not only leads to substantial mortality but also becomes a major challenge to effective treatment. Epithelial-mesenchymal transition (EMT) is a key mechanism facilitating cancer metastasis. Targeting the EMT process with more efficacious and less toxic agents to prevent metastasis is of significant therapeutic value for ovarian cancer treatment. The anti-EMT function and mechanism of ginsenoside Rb1, a monomer composition extracted from the traditional Chinese herb Panax ginseng or P. notoginseng , was investigated in the present study. Western blotting demonstrated that treatment with ginsenoside Rb1 antagonized hypoxia-induced E-cadherin downregulation and vimentin upregulation in SKOV3 and 3AO human ovarian cancer cells. Wound healing assays and in vitro migration assays indicated that ginsenoside Rb1 weakened hypoxia-enhanced cell migration ability. Furthermore, it was demonstrated that microRNA (miR)-25 is upregulated by hypoxia in ovarian cancer cells, which was attenuated by ginsenoside Rb1 treatment. Additionally, forced expression of miR-25 in ovarian cancer cells was identified to not only trigger EMT, but also block the suppressive effects of ginsenoside Rb1 on hypoxia-induced EMT by negatively targeting the E-cadherin transactivator, EP300. In conclusion, ginsenoside Rb1 may reverse hypoxia-induced EMT by abrogating the suppression of miR-25 on EP300 and E-cadherin, which suggests that ginsenoside Rb1 may be a potential therapeutic candidate for the treatment of ovarian cancer.

  8. HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

    Science.gov (United States)

    Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

    2015-01-01

    Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation.

    Science.gov (United States)

    Park, Yoon Shin; Kim, Gyungah; Jin, Yoon Mi; Lee, Jee Young; Shin, Jong Wook; Jo, Inho

    2016-01-08

    We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5'-deletions from -3040 to -1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Polina I. Bobyleva

    2016-01-01

    Full Text Available Human adipose tissue-stromal derived cells (ASCs are considered a perspective tool for regenerative medicine. Depending on the application mode ASC/allogeneic immune cell interaction can occur in the systemic circulation under plenty high concentrations of O2 and in target tissues at lower O2 levels. Here we examined the effects of allogeneic PHA-stimulated peripheral blood mononuclear cells (PBMCs on ASCs under ambient (20% oxygen and “physiological” hypoxia (5% O2. As revealed with microarray analysis ASCs under 20% O2 were more affected by activated PBMCs, which was manifested in differential expression of more than 300 genes, whereas under 5% O2 only 140 genes were changed. Altered gene pattern was only partly overlapped at different O2 conditions. Under O2 ASCs retained their proliferative and differentiative capacities, mesenchymal phenotype, and intracellular organelle’ state. ASCs were proinflammatory activated on transcription level that was confirmed by their ability to suppress activation and proliferation of mitogen-stimulated PBMCs. ASC/PBMCs interaction resulted in anti-inflammatory shift of paracrine mediators in conditioning medium with significant increase of immunosuppressive LIF level. Our data indicated that under both ambient and tissue-related O2 ASCs possessed immunosuppressive potential and maintained functional activity. Under “physiological” hypoxia ASCs were less susceptible to “priming” by allogeneic mitogen-activated PBMCs.

  11. Erythrocyte-Membrane-Enveloped Perfluorocarbon as Nanoscale Artificial Red Blood Cells to Relieve Tumor Hypoxia and Enhance Cancer Radiotherapy.

    Science.gov (United States)

    Gao, Min; Liang, Chao; Song, Xuejiao; Chen, Qian; Jin, Qiutong; Wang, Chao; Liu, Zhuang

    2017-09-01

    Hypoxia, a common feature within many types of solid tumors, is known to be closely associated with limited efficacy for cancer therapies, including radiotherapy (RT) in which oxygen is essential to promote radiation-induced cell damage. Here, an artificial nanoscale red-blood-cell system is designed by encapsulating perfluorocarbon (PFC), a commonly used artificial blood substitute, within biocompatible poly(d,l-lactide-co-glycolide) (PLGA), obtaining PFC@PLGA nanoparticles, which are further coated with a red-blood-cell membrane (RBCM). The developed PFC@PLGA-RBCM nanoparticles with the PFC core show rather efficient loading of oxygen, as well as greatly prolonged blood circulation time owing to the coating of RBCM. With significantly improved extravascular diffusion within the tumor mass, owing to their much smaller nanoscale sizes compared to native RBCs with micrometer sizes, PFC@PLGA-RBCM nanoparticles are able to effectively deliver oxygen into tumors after intravenous injection, leading to greatly relieved tumor hypoxia and thus remarkably enhanced treatment efficacy during RT. This work thus presents a unique type of nanoscale RBC mimic for efficient oxygen delivery into solid tumors, favorable for cancer treatment by RT, and potentially other types of therapy as well. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Blood outgrowth endothelial cells reduce hypoxia-mediated fibroblast to myofibroblast conversion by decreasing pro-angiogenic cytokines

    Science.gov (United States)

    Torres, Evelyn Nieves; Yang, Binxia; Brahmbhatt, Akshaar; Mukhopadhyay, Debabrata; Misra, Sanjay

    2015-01-01

    Purpose Hypoxic conditions cause fibroblasts to differentiate to a smooth muscle cell actin (α -SMA) positive cells (myofibroblasts), which is a hallmark of venous neointimal hyperplasia (VNH) associated with hemodialysis vascular access. The purpose of the present study was to determine if BOEC may reduce fibroblast to myofibroblast conversion under hypoxic conditions and what are the potential mechanisms. Methods An experimental model was used in which fibroblasts and BOEC were subjected to hypoxia under contact and transwell conditions to determine if BOEC reduce the conversion of fibroblasts to myofibroblasts under hypoxic conditions. Gene expression under different conditions was performed. In addition, functional assays including cell proliferation and migration were determined. Results This study demonstrates that contact needs to occur between BOEC and fibroblast for the reduction in hypoxia driven conversion of fibroblast to α-SMA. This is associated with a decrease in several pro-angiogenic genes including vascular endothelial growth factor (VEGF-A), platelet derived growth factor (PDGF), fibroblast growth factor (FGF-1), and matrix metalloproteinase-2 (MMP-2) in fibroblasts in contact with BOEC when compared to fibroblasts alone. In addition, migration is significantly reduced while proliferation remains unchanged. Conclusion This study helps provide rationale for using BOEC delivered to the adventitia of the outflow vein of hemodialysis vascular access to reduce VNH. PMID:25677750

  13. Influence of hypoxia on the domiciliation of Mesenchymal Stem Cells after infusion into rats: possibilities of targeting pulmonary artery remodeling via cells therapies?

    Directory of Open Access Journals (Sweden)

    Domenech Jorge

    2005-10-01

    Full Text Available Abstract Background Bone marrow (BM cells are promising tools for vascular therapies. Here, we focused on the possibility of targeting the hypoxia-induced pulmonary artery hypertension remodeling with systemic delivery of BM-derived mesenchymal stem cells (MSCs into non-irradiated rats. Methods Six-week-old Wistar rats were exposed to 3-week chronic hypoxia leading to pulmonary artery wall remodeling. Domiciliation of adhesive BM-derived CD45- CD73+ CD90+ MSCs was first studied after a single intravenous infusion of Indium-111-labeled MSCs followed by whole body scintigraphies and autoradiographies of different harvested organs. In a second set of experiments, enhanced-GFP labeling allowed to observe distribution at later times using sequential infusions during the 3-week hypoxia exposure. Results A 30% pulmonary retention was observed by scintigraphies and no differences were observed in the global repartition between hypoxic and control groups. Intrapulmonary radioactivity repartition was homogenous in both groups, as shown by autoradiographies. BM-derived GFP-labeled MSCs were observed with a global repartition in liver, in spleen, in lung parenchyma and rarely in the adventitial layer of remodeled vessels. Furthermore this global repartition was not modified by hypoxia. Interestingly, these cells displayed in vivo bone marrow homing, proving a preservation of their viability and function. Bone marrow homing of GFP-labeled MSCs was increased in the hypoxic group. Conclusion Adhesive BM-derived CD45- CD73+ CD90+ MSCs are not integrated in the pulmonary arteries remodeled media after repeated intravenous infusions in contrast to previously described in systemic vascular remodeling or with endothelial progenitor cells infusions.

  14. TCDD Induces the Hypoxia-Inducible Factor (HIF-1α Regulatory Pathway in Human Trophoblastic JAR Cells

    Directory of Open Access Journals (Sweden)

    Tien-Ling Liao

    2014-09-01

    Full Text Available The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K inhibitor or N-acetylcysteine (a ROS scavenger. The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ, PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

  15. Hypoxia precondition promotes adipose-derived mesenchymal stem cells based repair of diabetic erectile dysfunction via augmenting angiogenesis and neuroprotection.

    Directory of Open Access Journals (Sweden)

    XiYou Wang

    Full Text Available The aim of the present study was to examine whether hypoxia preconditioning could improve therapeutic effects of adipose derived mesenchymal stem cells (AMSCs for diabetes induced erectile dysfunction (DED. AMSCs were pretreated with normoxia (20% O2, N-AMSCs or sub-lethal hypoxia (1% O2, H-AMSCs. The hypoxia exposure up-regulated the expression of several angiogenesis and neuroprotection related cytokines in AMSCs, including vascular endothelial growth factor (VEGF and its receptor FIK-1, angiotensin (Ang-1, basic fibroblast growth factor (bFGF, brain-derived neurotrophic factor (BDNF, glial cell-derived neurotrophic factor (GDNF, stromal derived factor-1 (SDF-1 and its CXC chemokine receptor 4 (CXCR4. DED rats were induced via intraperitoneal injection of streptozotocin (60 mg/kg and were randomly divided into three groups-Saline group: intracavernous injection with phosphate buffer saline; N-AMSCs group: N-AMSCs injection; H-AMSCs group: H-AMSCs injection. Ten rats without any treatment were used as normal control. Four weeks after injection, the mean arterial pressure (MAP and intracavernosal pressure (ICP were measured. The contents of endothelial, smooth muscle, dorsal nerve in cavernoursal tissue were assessed. Compared with N-AMSCs and saline, intracavernosum injection of H-AMSCs significantly raised ICP and ICP/MAP (p<0.05. Immunofluorescent staining analysis demonstrated that improved erectile function by MSCs was significantly associated with increased expression of endothelial markers (CD31 and vWF (p<0.01 and smooth muscle markers (α-SMA (p<0.01. Meanwhile, the expression of nNOS was also significantly higher in rats receiving H-AMSCs injection than those receiving N-AMSCs or saline injection. The results suggested that hypoxic preconditioning of MSCs was an effective approach to enhance their therapeutic effect for DED, which may be due to their augmented angiogenesis and neuroprotection.

  16. Low Cancer Stem Cell Marker Expression and Low Hypoxia Identify Good Prognosis Subgroups in HPV(-) HNSCC after Postoperative Radiochemotherapy: A Multicenter Study of the DKTK-ROG

    DEFF Research Database (Denmark)

    Linge, Annett; Löck, Steffen; Gudziol, Volker

    2016-01-01

    PURPOSE: To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: Expressio...

  17. Galectin-3 induced by hypoxia promotes cell migration in thyroid cancer cells.

    Science.gov (United States)

    Zheng, Jiaojiao; Lu, Weihui; Wang, Cong; Xing, Yang; Chen, Xiaoning; Ai, Zhilong

    2017-11-24

    The aim of this study is to investigate the role of Galectin-3 in human thyroid cancer migration. The expression of Galectin-3 in surgical specimens was investigated using immunohistochemistry and western blot. A papillary thyroid cancer cell line (B-cpap) and an anaplastic thyroid cancer cell line (8305c) were transfected with short-hairpin RNA against Galectin-3 (Gal-3-shRNA). Low-molecular citrus pectin (LCP) was also used to antagonize Galectin-3. The migration and invasion of the cell lines were examined. The related signaling pathways were investigated to explore the Galectin-3 mechanism of action. Galectin-3 was highly expressed in metastasized thyroid cancers. Knocking down and antagonizing Galectin-3 significantly suppressed the migration of thyroid cancer cells. Knocking down Galectin-3 inhibited the activity of Wnt, MAPK, Src and Rho signaling pathways. Galectin-3 was up-regulated via HIF-1α in a hypoxic environment. Galectin-3 knockdown could reduce cell motility in hypoxic environments. This study suggests that Galectin-3 could act as a modulator of thyroid cancer migration, especially in hypoxic microenvironments. This regulation function of Galectin-3 may work through multiple signaling pathways.

  18. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  19. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    International Nuclear Information System (INIS)

    Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2012-01-01

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl 2 ), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl 2 treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl 2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl 2 -induced reactive oxygen species (ROS) formation and completely negated CoCl 2 -induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl 2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl 2 increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl 2 -induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical

  20. Survival of hypoxic human mesenchymal stem cells is enhanced by a positive feedback loop involving miR-210 and hypoxia-inducible factor 1.

    Science.gov (United States)

    Chang, Woochul; Lee, Chang Youn; Park, Jun-Hee; Park, Moon-Seo; Maeng, Lee-So; Yoon, Chee Soon; Lee, Min Young; Hwang, Ki-Chul; Chung, Yong-An

    2013-01-01

    The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1α activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1α. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1α expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1α was important for MSC survival under hypoxic conditions.

  1. Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Suna, E-mail: wangs3@mail.nih.gov; Zhou, Yifu; Andreyev, Oleg; Hoyt, Robert F.; Singh, Avneesh; Hunt, Timothy; Horvath, Keith A.

    2014-04-15

    Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions.

  2. Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

    International Nuclear Information System (INIS)

    Wang, Suna; Zhou, Yifu; Andreyev, Oleg; Hoyt, Robert F.; Singh, Avneesh; Hunt, Timothy; Horvath, Keith A.

    2014-01-01

    Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative RT PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions.

  3. Baicalin Inhibits Hypoxia-Induced Pulmonary Artery Smooth Muscle Cell Proliferation via the AKT/HIF-1α/p27-Associated Pathway

    Directory of Open Access Journals (Sweden)

    Lin Zhang

    2014-05-01

    Full Text Available Baicalin, a flavonoid compound purified from the dry roots of Scutellaria baicalensis Georgi, has been shown to possess various pharmacological actions. Previous studies have revealed that baicalin inhibits the growth of cancer cells through the induction of apoptosis. Pulmonary arterial hypertension (PAH is a devastating disease characterized by enhanced pulmonary artery smooth muscle cell (PASMCs proliferation and suppressed apoptosis. However, the potential mechanism of baicalin in the regulation of PASMC proliferation and the prevention of cardiovascular diseases remains unexplored. To test the effects of baicalin on hypoxia, we used rats treated with or without baicalin (100 mg·kg−1 each rat at the beginning of the third week after hypoxia. Hemodynamic and pulmonary pathomorphology data showed that right ventricular systolic pressures (RVSP, the weight of the right ventricle/left ventricle plus septum (RV/LV + S ratio and the medial width of pulmonary arterioles were much higher in chronic hypoxia. However, baicalin treatment repressed the elevation of RVSP, RV/LV + S and attenuated the pulmonary vascular structure remodeling (PVSR of pulmonary arterioles induced by chronic hypoxia. Additionally, baicalin (10 and 20 μmol·L−1 treatment suppressed the proliferation of PASMCs and attenuated the expression of hypoxia-inducible factor-α (HIF-α under hypoxia exposure. Meanwhile, baicalin reversed the hypoxia-induced reduction of p27 and increased AKT/protein kinase B phosphorylation p-AKT both in vivo and in vitro. These results suggested that baicalin could effectively attenuate PVSR and hypoxic pulmonary hypertension.

  4. Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells

    Directory of Open Access Journals (Sweden)

    Dai Zhi-Jun

    2012-03-01

    Full Text Available Abstract Background The exact mechanism of the effects of hypoxia on the proliferation and apoptosis in carcinoma cells is still conflicting. This study investigated the variation of hypoxia-inducible factor-1α(HIF-1α expression and the apoptosis effect of hypoxia stimulated by cobalt chloride (CoCl2 in pancreatic cancer PC-2 cells. Methods PC-2 cells were cultured with different concentration (50-200 μmol/L of CoCl2 after 24-120 hours to simulate hypoxia in vitro. The proliferation of PC-2 cells was examined by MTT assay. The cellular morphology of PC-2 cells were observed by light inverted microscope and transmission electron microscope(EM. The expression of HIF-1α on mRNA and protein level was measured by semi-quantitive RT-PCR and Western blot analysis. Apoptosis of PC-2 cells were demonstrated by flow cytometry with Annexin V-FITC/PI double staining. Results MTT assay showed that the proliferation of PC-2 cells were stimulated in the first 72 h, while after treated over 72 h, a dose- dependent inhibition of cell growth could be observed. By using transmission electron microscope, swollen chondrosomes, accumulated chromatin under the nuclear membrane and apoptosis bodies were observed. Flow cytometer(FCM analysis showed the apoptosis rate was correlated with the dosage of CoCl2. RT-PCR and Western blot analysis indicated that hypoxia could up-regulate the expression of HIF-1α on both mRNA and protein levels. Conclusion Hypoxic microenvironment stimulated by CoCl2 could effectively induce apoptosis and influence cell proliferation in PC-2 cells, the mechanism could be related to up-expression of HIF-1α.

  5. The influence of the hypoxia on bio electric activity of myocardium cells after acute gamma-irradiation

    International Nuclear Information System (INIS)

    Malykhina, A.P.; Lobanok, L.M.

    1998-01-01

    The aim of the investigation was to evaluate bio electric activity of the heart after acute gamma-irradiation and the hypoxia. Female rats (5 - 6 month old) was acute irradiated at 0,5 Gy dose (dose rate 0,1 mGy/sec) and was examined in a 10, 30, 90 and 180 days later. Electrophysiological study of the isolated right rat auricle was conducted by means of microelectrode registration of intracellular bio electric activity. It was shown that gamma-irradiation at 0,5 Gy resulted in decrease of amplitude and duration of action potentials of right auricle cells, attenuation of the dependence of the electrophysiological characteristics upon the stimulation frequency. Exposure of the rats to acute gamma-irradiation leaded to the depression of the interval of the cardio myocyte reaction on the hypoxia. The revealed post-radiation changes determined the reduction in function resistance of heart cells bio electric activity and can promote the initiation of arrhythmias

  6. A3 Adenosine Receptors Modulate Hypoxia-inducible Factor-1a Expression in Human A375 Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Stefania Merighi

    2005-10-01

    Full Text Available Hypoxia-inducible factor-1 (HIF-1 is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% 02, adenosine upregulates HIF-1α protein expression in a dose-dependent and timedependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2 protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1a and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.

  7. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis.

    Science.gov (United States)

    Fan, Lihong; Zhang, Chen; Yu, Zefeng; Shi, Zhibin; Dang, Xiaoqian; Wang, Kunzheng

    2015-12-01

    Osteonecrosis of the femoral head may be a disease resulting from abnormal proliferation or differentiation of mesenchymal stem cells. The present investigation explored the novel strategy of hypoxia-preconditioned BMMSCs to reverse the impairment of osteonecrosis BMMSCs and enhance the therapeutic potential of hypoxia-treated BMMSC transplantation. BMMSCs from the anterior superior iliac spine region of osteonecrosis rabbit were cultured under 20% O2 or 2% O2 conditions. Normal BMMSCs were cultured under 20% O2 condition as control. Growth factors secreted were examined by enzyme-linked immunosorbent assay. 20% O2 or 2% O2 BMMSCs were injected into the femoral head of rabbits after core decompression. Cell viability and apoptosis were assessed in vitro, and TUNEL staining of the femoral head was analyzed after transplantation. Angiogenesis (capillary-like structure formation, CD31 immunohistochemical staining and ink infusion angiography) and osteogenesis (Alizarin red-S staining, micro-CT scanning and OCN immunohistochemical staining) tests were conducted as well. 2% O2 exposure up-regulated growth factor secretion in BMMSCs. Apoptosis in 2% O2 group was lower when compared with that in 20% O2 osteonecrosis group. Cell viability in 2% O2 was significantly higher when compared with that in 20% O2 osteonecrosis group. Growth factor secretion, cell viability, apoptosis, capillary-like structure formation, Alizarin red-S staining, and ALP staining showed no difference between the 2% O2 BMMSC and normal BMMSC groups. Transplantation of 2% O2 versus 20% O2 mesenchymal stem cells after core decompression resulted in an increase in angiogenesis function and a decrease in local tissue apoptosis. Our study also found that osteogenesis function was improved after hypoxic stem cell transplantation. Hypoxic preconditioning of BMMSCs is an effective means of reversing the impairment of osteonecrosis BMMSCs, promoting their regenerative capability and therapeutic potential for

  8. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA.

    Directory of Open Access Journals (Sweden)

    Mari Gotoh

    Full Text Available Cyclic phosphatidic acid (cPA is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2 to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A cells with CoCl(2 induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2. Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1, LPA(2, and LPA(6, which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1 and LPA(2 have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1 and LPA(2 on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1 and LPA(3 antagonist, was adopted to know the LPA(1 function and siRNA was used to knockdown the expression of LPA(2. On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2-induced hypoxia damage is mediated via LPA(2.

  9. The Proteasome Inhibitor MG-132 Protects Hypoxic SiHa Cervical Carcinoma Cells after Cyclic Hypoxia/Reoxygenation from Ionizing Radiation

    Directory of Open Access Journals (Sweden)

    Frank Pajonk

    2006-12-01

    Full Text Available INTRODUCTION: Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of radiation therapy. This study was designed to determine how varying cycles of hypoxia/reoxygenation affect the response of cervical carcinoma cells irradiated under oxic and hypoxic conditions and whether this could be modulated by proteasome inhibition. MATERIALS AND METHODS: Plateau-phase SiHa cervical carcinoma cells in culture were exposed to varying numbers of 30-minute cycles of hypoxia/reoxygenation directly before irradiation under oxic or hypoxic conditions. 26S Proteasome activity was blocked by addition of MG-132. Clonogenic survival was measured by a colonyforming assay. RESULTS: Under oxic conditions, repeated cycles of hypoxia/reoxygenation decreased the clonogenic survival of SiHa cells. This effect was even more pronounced after the inhibition of 26S proteasome complex. In contrast, under hypoxic conditions, SiHa cells were radioresistant, as expected, but this was increased by proteasome inhibition. CONCLUSIONS: Proteasome inhibition radiosensitizes oxygenated tumor cells but may also protect tumor cells from ionizing radiation under certain hypoxic conditions.

  10. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  11. Human TM9SF4 Is a New Gene Down-Regulated by Hypoxia and Involved in Cell Adhesion of Leukemic Cells.

    Directory of Open Access Journals (Sweden)

    Rosa Paolillo

    Full Text Available The transmembrane 9 superfamily protein member 4, TM9SF4, belongs to the TM9SF family of proteins highly conserved through evolution. TM9SF4 homologs, previously identified in many different species, were mainly involved in cellular adhesion, innate immunity and phagocytosis. In human, the function and biological significance of TM9SF4 are currently under investigation. However, TM9SF4 was found overexpressed in human metastatic melanoma and in a small subset of acute myeloid leukemia (AMLs and myelodysplastic syndromes, consistent with an oncogenic function of this gene.In this study, we first analyzed the expression and regulation of TM9SF4 in normal and leukemic cells and identified TM9SF4 as a gene highly expressed in human quiescent CD34+ hematopoietic progenitor cells (HPCs, regulated during monocytic and granulocytic differentiation of HPCs, both lineages giving rise to mature myeloid cells involved in adhesion, phagocytosis and immunity. Then, we found that TM9SF4 is markedly overexpressed in leukemic cells and in AMLs, particularly in M2, M3 and M4 AMLs (i.e., in AMLs characterized by the presence of a more or less differentiated granulocytic progeny, as compared to normal CD34+ HPCs. Proliferation and differentiation of HPCs occurs in hypoxia, a physiological condition in bone marrow, but also a crucial component of cancer microenvironment. Here, we investigated the impact of hypoxia on TM9SF4 expression in leukemic cells and identified TM9SF4 as a direct target of HIF-1α, downregulated in these cells by hypoxia. Then, we found that the hypoxia-mediated downregulation of TM9SF4 expression is associated with a decrease of cell adhesion of leukemic cells to fibronectin, thus demonstrating that human TM9SF4 is a new molecule involved in leukemic cell adhesion.Altogether, our study reports for the first time the expression of TM9SF4 at the level of normal and leukemic hematopoietic cells and its marked expression at the level of AMLs

  12. Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells

    Directory of Open Access Journals (Sweden)

    JV Stoyanov

    2011-06-01

    Full Text Available There is evidence that mesenchymal stem cells (MSCs can differentiate towards an intervertebral disc (IVD-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß to the effects of hypoxia, growth and differentiation factor-5 (GDF5, and coculture with bovine nucleus pulposus cells (bNPC. The efficacy of molecules recently discovered as possible nucleus pulposus (NP markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 % or normal (20 % oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

  13. Inhibition of phospholipaseD2 increases hypoxia-induced human colon cancer cell apoptosis through inactivating of the PI3K/AKT signaling pathway.

    Science.gov (United States)

    Liu, Maoxi; Fu, Zhongxue; Wu, Xingye; Du, Kunli; Zhang, Shouru; Zeng, Li

    2016-05-01

    Hypoxia is a common feature of solid tumor, and is a direct stress that triggers apoptosis in many human cell types. As one of solid cancer, hypoxia exists in the whole course of colon cancer occurrence and progression. Our previous studies shown that hypoxia induce high expression of phospholipase D2 (PLD2) and survivin in colon cancer cells. However, the correlation between PLD2 and survivin in hypoxic colon cancer cells remains unknown. In this study, we observed significantly elevated PLD2 and survivin expression levels in colon cancer tissues and cells. This is a positive correlation between of them, and co-expression of PLD2 and survivin has a positive correlation with the clinicpatholic features including tumor size, TNM stage, and lymph node metastasis. We also found that hypoxia induced the activity of PLD increased significant mainly caused by PLD2 in colon cancer cells. However, inhibition the activity of PLD2 induced by hypoxia promotes the apoptosis of human colon cancer cells, as well as decreased the expression of apoptosis markers including survivin and bcl2. Moreover, the pharmacological inhibition of PI3K/AKT supported the hypothesis that promotes the apoptosis of hypoxic colon cancer cells by PLD2 activity inhibition may through inactivation of the PI3K/AKT signaling pathway. Furthermore, interference the PLD2 gene expression leaded to the apoptosis of hypoxic colon cancer cells increased and also decreased the expression level of survivin and bcl2 may through inactivation of PI3K/AKT signaling pathway. These results indicated that PLD2 play antiapoptotic role in colon cancer under hypoxic conditions, inhibition of the activity, or interference of PLD2 gene expression will benefit for the treatment of colon cancer patients.

  14. Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.

    Science.gov (United States)

    Ciapetti, Gabriela; Granchi, Donatella; Fotia, Caterina; Savarino, Lucia; Dallari, Dante; Del Piccolo, Nicola; Donati, Davide Maria; Baldini, Nicola

    2016-09-01

    Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1-3% oxygen tension, the survival and function of multipotent cells is questionable. In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays. Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension. These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. Heat shock protein 90β inhibits apoptosis of intestinal epithelial cells induced by hypoxia through stabilizing phosphorylated Akt

    Directory of Open Access Journals (Sweden)

    Shuai Zhang

    2013-01-01

    Full Text Available Intestinal epithelial cell (IEC apoptosis induced by hypoxiacompromise intestinal epithelium barrier function. Both Akt andHsp90 have cytoprotective function. However, the specific roleof Akt and Hsp90β in IEC apoptosis induced by hypoxia has notbeen explored. We confirmed that hypoxia-induced apoptosiswas reduced by Hsp90β overexpression but enhanced bydecreasing Hsp90β expression. Hsp90β overexpressionenhanced BAD phosphorylation and thus reduced mitochondrialrelease of cytochrome C. Reducing Hsp90β expression hadopposite effects. The protective effect of Hsp90β againstapoptosis was negated by LY294002, an Akt inhibitor. Furtherstudy showed that Akt phosphorylation was enhanced byHsp90β, which was not due to the activation of upstream PI3Kand PDK1 but because of stabilization of pAkt via directinteraction between Hsp90β and pAkt. These results demonstratethat Hsp90β may play a significant role in protecting IECs fromhypoxia-induced apoptosis via stabilizing pAkt to phosphorylateBAD and reduce cytochrome C release. [BMB Reports 2013;46(1: 47-52

  16. Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis through the Akt and PERK Pathways

    Directory of Open Access Journals (Sweden)

    Pingping Fu

    2016-11-01

    Full Text Available Retinal ganglion cells (RGCs consume large quantities of energy to convert light information into a neuronal signal, which makes them highly susceptible to hypoxic injury. This study aimed to investigate the potential protection by baclofen, a GABAB receptor agonist, of retinal ganglion cells against hypoxia-induced apoptosis. CoCl2 was applied to mimic hypoxia. Primary rat retinal ganglion cells (RGCs were subjected to CoCl2 with or without baclofen treatment, and RNA interference techniques were used to knock down the GABAB2 gene in the primary RGCs. The viability and apoptosis of RGCs were assessed using cell viability and TUNEL assays, Hoechst staining, and flow cytometry. The expression of cleaved caspase-3, bcl-2, bax, Akt, phospho-Akt, PERK, phospho-PERK, eIF2α, phospho-eIF2α, ATF-4, and CHOP were measured using western blotting. GABAB2 mRNA expression was determined using quantitative real-time polymerase chain reaction (qRT-PCR analysis. Our study revealed that CoCl2 significantly induced RGC apoptosis and that baclofen reversed these effects. CoCl2-induced reduction of Akt activity was also reversed by baclofen. Baclofen prevented the activation of the PERK pathway and the increase in CHOP expression induced by CoCl2. Knockdown of GABAB2 and the inactivation of the Akt pathway by inhibitors reduced the protective effect of baclofen on CoCl2-treated RGCs. Taken together, these results demonstrate that baclofen protects RGCs from CoCl2-induced apoptosis by increasing Akt activity and by suppressing the PERK pathway and CHOP activation.

  17. Effects of bone marrow-derived cells on monocrotaline- and hypoxia-induced pulmonary hypertension in mice

    Directory of Open Access Journals (Sweden)

    Vainchenker William

    2007-01-01

    Full Text Available Abstract Background Bone marrow -derived cells (BMDCs can either limit or contribute to the process of pulmonary vascular remodeling. Whether the difference in their effects depends on the mechanism of pulmonary hypertension (PH remains unknown. Objectives We investigated the effect of BMDCs on PH induced in mice by either monocrotaline or exposure to chronic hypoxia. Methods Intravenous administration of the active monocrotaline metabolite (monocrotaline pyrrole, MCTp to C57BL/6 mice induced PH within 15 days, due to remodeling of small distal vessels. Three days after the MCTp injection, the mice were injected with BMDCs harvested from femurs and tibias of donor mice treated with 5-fluorouracil (3.5 mg IP/animal to deplete mature cells and to allow proliferation of progenitor cells. Results BMDCs significantly attenuated PH as assessed by reductions in right ventricular systolic pressure (20 ± 1 mmHg vs. 27 ± 1 mmHg, P ≤ 0.01, right ventricle weight/left ventricle+septum weight ratio (0.29 ± 0.02 vs. 0.36 ± 0.01, P ≤ 0.03, and percentage of muscularized vessels (26.4% vs. 33.5%, P ≤ 0.05, compared to control animals treated with irradiated BMDCs. Tracking cells from constitutive GFP-expressing male donor mice with anti-GFP antibodies or chromosome Y level measurement by quantitative real-time PCR showed BMDCs in the lung. In contrast, chronically hypoxic mice subjected to the same procedure failed to show improvement in PH. Conclusion These results show that BMDCs limit pulmonary vascular remodeling induced by vascular injury but not by hypoxia.

  18. Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

    2011-04-15

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  19. Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    International Nuclear Information System (INIS)

    Staab, Adrian; Fleischer, Markus; Wuerzburg Univ.; Loeffler, Juergen; Einsele, Herrmann; Said, Harun M.; Katzer, Astrid; Flentje, Michael; Plathow, Christian; Vordermark, Dirk; Halle-Wittenberg Univ.

    2011-01-01

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O 2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O 2 as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  20. Antihypoxic effect of miR-24 in SH-SY5Y cells under hypoxia via downregulating expression of neurocan

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xingyuan, E-mail: sunxingyuan@sina.com; Ren, Zhanjun; Pan, Yunzhi; Zhang, Chenxin

    2016-09-02

    Hypoxia-induced apoptosis-related mechanisms involved in the brain damage following cerebral ischemia injury. A subset of the small noncoding microRNA (miRNAs) is regulated by tissue oxygen levels, and miR-24 was found to be activated by hypoxic conditions. However, the roles of miR-24 and its target gene in neuron are not well understood. Here, we validated miRNA-24 is down-regulated in patients with cerebral infarction. Hypoxia suppressed the expression of miR-24, but increased the expression of neurocan in both mRNA and protein levels in SH-SY5Y cells. MiR-24 mimics reduced the expression of neurocan, suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. By luciferase reporter assay, neurocan is validated a direct target gene of miR-24. Furthermore, knockdown of neurocan suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. Taken together, miR-24 overexpression or silencing of neurocan shows an antihypoxic effect in SH-SY5Y cells. Therefore, miR-24 and neurocan play critical roles in neuron cell apoptosis and are potential therapeutic targets for ischemic brain disease. - Highlights: • miR-24 and neurocan play critical roles in neuron cell apoptosis. • miR-24 and neurocan are potential therapeutic targets for ischemic brain disease. • Antihypoxic effect of miR-24 and neurocan in SH-SY5Y cells.

  1. Effect of transplants of retinal pigment epithelial cells from adult human eye on degenerative processes in the brain of rats with experimental acute hypoxia.

    Science.gov (United States)

    Aleksandrova, M A; Kuznetsova, A V; Verdiev, B I; Milyushina-Rzhanova, L A; Sukhinich, K K

    2014-05-01

    Stimulation of cell regeneration in the brain and eye retina in various degenerative processes is a pressing problem in neurobiology. A promising approach is transplantation of somatic cells reprogrammed towards neural lineage. We studied the effect of transplantation of retinal pigment epithelial cells from adult human eye transdifferentiated in culture on degenerative processes in the brain of rats subjected to acute hypoxia. Immunohistochemical and molecular genetic analysis suggests that retinal pigment epithelial cells transdifferentiate in vitro and express markers of low-differentiated neural cells. The cells transplanted into rat brain survive for at least 20 days. During this period, they stimulate compensatory and reparative processes that protected cortical neurons in the recipients from hypoxia-induced degeneration.

  2. Cordyceps sinensis increases hypoxia tolerance by inducing heme oxygenase-1 and metallothionein via Nrf2 activation in human lung epithelial cells.

    Science.gov (United States)

    Singh, Mrinalini; Tulsawani, Rajkumar; Koganti, Praveen; Chauhan, Amitabh; Manickam, Manimaran; Misra, Kshipra

    2013-01-01

    Cordyceps sinensis, an edible mushroom growing in Himalayan regions, is widely recognized in traditional system of medicine. In the present study, we report the efficacy of Cordyceps sinensis in facilitating tolerance to hypoxia using A549 cell line as a model system. Treatment with aqueous extract of Cordyceps sinensis appreciably attenuated hypoxia induced ROS generation, oxidation of lipids and proteins and maintained antioxidant status similar to that of controls via induction of antioxidant gene HO1 (heme oxygenase-1), MT (metallothionein) and Nrf2 (nuclear factor erythroid-derived 2-like 2). In contrast, lower level of NF κ B (nuclear factor kappaB) and tumor necrosis factor- α observed which might be due to higher levels of HO1, MT and transforming growth factor- β . Further, increase in HIF1 (hypoxia inducible factor-1) and its regulated genes; erythropoietin, vascular endothelial growth factor, and glucose transporter-1 was observed. Interestingly, Cordyceps sinensis treatment under normoxia did not regulate the expression HIF1, NF κ B and their regulated genes evidencing that Cordyceps sinensis per se did not have an effect on these transcription factors. Overall, Cordyceps sinensis treatment inhibited hypoxia induced oxidative stress by maintaining higher cellular Nrf2, HIF1 and lowering NF κ B levels. These findings provide a basis for possible use of Cordyceps sinensis in tolerating hypoxia.

  3. Inhibition of hypoxia-inducible factor 1 with acriflavine sensitizes hypoxic tumor cells to photodynamic therapy with zinc phthalocyanine-encapsulating cationic liposomes

    NARCIS (Netherlands)

    Broekgaarden, Mans; Weijer, Ruud; Krekorian, Massis; van den IJssel, Bas; Kos, Milan; Alles, Lindy K.; van Wijk, Albert C.; Bikadi, Zsolt; Hazai, Eszter; van Gulik, Thomas M.; Heger, Michal

    2016-01-01

    Photodynamic therapy (PDT) is a tumor treatment modality in which a tumorlocalized photosensitizer is excited with light, which results in local production of reactive oxygen species, destruction of tumor vasculature, tumor hypoxia, tumor cell death, and induction of an anti-tumor immune response.

  4. Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response

    NARCIS (Netherlands)

    Nagelkerke, A.; Bussink, J.; Mujcic, H.; Wouters, B.G.; Lehmann, S.A.; Sweep, F.C.; Span, P.N.

    2013-01-01

    ABSTRACT: INTRODUCTION: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like

  5. Hypoxia-inducible factor 1-alpha up-regulates the expression of phospholipase D2 in colon cancer cells under hypoxic conditions.

    Science.gov (United States)

    Liu, Maoxi; Du, Kunli; Fu, Zhongxue; Zhang, Shouru; Wu, Xingye

    2015-01-01

    Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that phospholipase D2 (PLD2) plays significant roles in cancer progression. In this study, correlation between the expression of PLD2 and the change in the protein level of hypoxia-inducible factor 1-alpha (HIF1-α) was studied. Thirty human colon cancer tissues were examined for the expression of HIF1-α and PLD2 protein, and mRNA levels. SW480 and SW620 cells were exposed to normoxia (20 %) or hypoxia (Hypoxic stress induced PLD2 mRNA and protein expression in SW480 and SW620 cells. Cells transfected with HIF1-α siRNA showed attenuation of hypoxia stress-induced PLD2 expression. In vivo growth decreased in response to HIF1-α and PLD2 inhibition. These results suggest that PLD2 expression in colon cancer cells is up-regulated via HIF1-α in response to hypoxic stress and underscores the crucial role of HIF1-α-induced PLD2 in tumor growth.

  6. Proteomics analysis of 3 different strains of Mycobacterium tuberculosis under in vitro hypoxia and evaluation of hypoxia associated antigen’s specific memory T cells in healthy household contacts

    Directory of Open Access Journals (Sweden)

    Santhi Devasundaram

    2016-09-01

    Full Text Available In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10 of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia experimental model. Cytosolic proteins were prepared from the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC and active pulmonary tuberculosis patients (PTB. Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p<0.005 for ESAT-6 and p<0.0005 for Lpd. This shows proteins that are predicted to be upregulated during in vitro hypoxia in most prevalent clinical strains would bring the possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also bring the probable true representative antigens that involved during adaption mechanism.

  7. Intermittent hypoxia hypobaric exposure minimized oxidative stress and antioxidants in brain cells of Sprague Dawleymice

    Directory of Open Access Journals (Sweden)

    Wardaya Wardaya

    2013-05-01

    Full Text Available AbstrakLatar belakang: Hipoksia hypobaric meningkatkan produksi radikal bebas, terutama spesies oksigen reaktif (ROS. Peningkatan ROS akan menyebabkan stres oksidatif bila tidak disertai dengan peningkatan enzim antioksidan. Kondisi ini dapat dikurangi dengan hipoksia hipobarik intermiten (HHI. Tujuan penelitian ini mengidentifikasi frekuensi IHH yang dapat meminimalkan efek hipoksia hipobarik terhadap stres oksidatif dan aktivitas antioksidan spesifik pada tikus Sprague Dawley.Metode: Penelitian eksperimental pada bulan Februari-April 2010, Subjek terdiri dari satu kelompok kontrol dan empat kelompok paparan pada mencit jantan Sprague Dawley. Setiap kelompok terdiri dari 5 tikus. Kelompok kontrol tidak terpapar IHH. Kelompok terpapar (dengan selang waktu satu minggu terpapar sekali, dua kali, tiga kali, atau empat kali IHH. Semua kelompok paparan dipaparkan hipobarik setara dengan ketinggian: 35.000 ft (1 menit, 25.000 ft (5 menit, dan 18.000 ft (25 menit. Jaringan otak diperiksa untuk 8-OHdG dan SOD.Hasil:Setelah tiga paparan IHH tingkat 8-OHdG sudah kembali ke nilai kontrol (P = 0,843. Tingkat SOD meningkat secara progresif pada dua, tiga, dan empat kali paparan IHH. Bahkan setelah paparan kedua, tingkat SOD sudah sama dengan nilai kontrol, 0,231 ± 0,042 (P = 0,191.Kesimpulan: Tiga kali IHH sudah dapat meminimalkan pengaruh hipoksia hipobarik terhadap stres oksidatif dan aktivitas spesifik antioksidan pada tikus Sprague Dawley.Kata kunci: hipoksia hipobarik intermiten, stres oksidatif, antioksidanAbstractBackground: Hypoxia hypobaric increase the production of free radicals, especially reactive oxygen species (ROS. The increase in ROS would cause oxidative stress when not accompanied by an increase in antioxidant enzymes. This condition may minimize by intermittent hypobaric hypoxia (IHH. This study aimed to identify the number of IHH which may minimize the effect of hypoxia hypobaric on oxidative stress and the specific activity of

  8. Urothelial carcinoma associated 1 is a hypoxia-inducible factor-1α-targeted long noncoding RNA that enhances hypoxic bladder cancer cell proliferation, migration, and invasion.

    Science.gov (United States)

    Xue, Mei; Li, Xu; Li, Zhengkun; Chen, Wei

    2014-07-01

    Urothelial carcinoma associated 1 (UCA1) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in bladder cancer progression and acts as a diagnostic biomarker for bladder carcinoma. Here, we studied the expression and function of lncRNA-UCA1 in the hypoxic microenvironment of bladder cancer. The expression and transcriptional activity of lncRNA-UCA1 were measured by quantitative real-time polymerase chain reaction and luciferase assays. Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry. Cell migration and invasion were detected by wound healing, migration, and invasion assays. The binding of hypoxia-inducible factor-1α (HIF-1α) to hypoxia response elements (HREs) in the lncRNA-UCA1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. HRE mutations were generated by using a site-directed mutagenesis kit, and HIF-1α knockdown was mediated by small interfering RNA. The effect of HIF-1α inhibition by YC-1 on lncRNA-UCA1 expression was also examined. LncRNA-UCA1 was upregulated by hypoxia in bladder cancer cells. Under hypoxic conditions, lncRNA-UCA1 upregulation increased cell proliferation, migration, and invasion and inhibited apoptosis. The underlying mechanism of hypoxia-upregulated lncRNA-UCA1 expression was that HIF-1α specifically bound to HREs in the lncRNA-UCA1 promoter. Furthermore, HIF-1α knockdown or inhibition could prevent lncRNA-UCA1 upregulation under hypoxia. These findings revealed the mechanism of lncRNA-UCA1 upregulation in hypoxic bladder cancer cells and suggested that effective blocking of lncRNA-UCA1 expression in the hypoxic microenvironment of bladder cancer could be a novel therapeutic strategy.

  9. Neuronal hypoxia in vitro: Investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells

    Directory of Open Access Journals (Sweden)

    Emmrich Frank

    2008-09-01

    Full Text Available Abstract Background The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood. Methods A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC, a stem cell enriched fraction (CD133+, 97.38% CD133-positive cells and a stem cell depleted fraction (CD133-, 0.06% CD133-positive cells of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y. Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9 and growth factors (G-CSF, GM-CSF, VEGF, bFGF were analyzed using fluorescence microscopy, FACS and cytometric bead array. Results tMNC, CD133+ and surprisingly CD133- reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% ± 3%. Untreated post-hypoxic control cultures showed apoptosis rates of 85% ± 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133- reduced apoptosis comparably. CD133- produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133+ cells were not detectable in direct co-cultures. Conclusion Our data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes.

  10. Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma.

    Directory of Open Access Journals (Sweden)

    Tjinta Brinkhuizen

    Full Text Available BACKGROUND: Basal cell carcinoma (BCC is the most common cancer in Caucasians. Trichoepithelioma (TE is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1 and mechanistic/mammalian target of rapamycin (mTOR are key players in these pathways. OBJECTIVES: To determine whether HIF1/mTOR signalling is involved in BCC and TE. METHODS: We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45 and TE (n = 35 samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, 80%. RESULTS: Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3, 73% and 75% (CAIX, 79% and 86% (GLUT1, 50% and 19% (HIF1α, 89% and 88% (pAKT, 55% and 61% (pS6, 15% and 25% (pMTOR, 44% and 63% (PHD2 and 44% and 49% (VEGF-A. CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included. CONCLUSIONS: HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE.

  11. Immunohistochemical Analysis of the Mechanistic Target of Rapamycin and Hypoxia Signalling Pathways in Basal Cell Carcinoma and Trichoepithelioma

    Science.gov (United States)

    Brinkhuizen, Tjinta; Weijzen, Chantal A. H.; Eben, Jonathan; Thissen, Monique R.; van Marion, Ariënne M.; Lohman, Björn G.; Winnepenninckx, Véronique J. L.; Nelemans, Patty J.; van Steensel, Maurice A. M.

    2014-01-01

    Background Basal cell carcinoma (BCC) is the most common cancer in Caucasians. Trichoepithelioma (TE) is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF) tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1) and mechanistic/mammalian target of rapamycin (mTOR) are key players in these pathways. Objectives To determine whether HIF1/mTOR signalling is involved in BCC and TE. Methods We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45) and TE (n = 35) samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, 80%). Results Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3), 73% and 75% (CAIX), 79% and 86% (GLUT1), 50% and 19% (HIF1α), 89% and 88% (pAKT), 55% and 61% (pS6), 15% and 25% (pMTOR), 44% and 63% (PHD2) and 44% and 49% (VEGF-A). CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included. Conclusions HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE. PMID:25181405

  12. Role of LncRNA MALAT-1 in hypoxia-induced PC12 cell injury via regulating p38MAPK signaling pathway.

    Science.gov (United States)

    Yang, Lin; Xu, Fei; Zhang, Miao; Shang, Xiao-Ying; Xie, Xin; Fu, Tao; Li, Jian-Ping; Li, Hong-Lin

    2018-03-23

    To investigate the role of LncRNA MALAT-1 in hypoxia-induced cell injury. Pheochromocytoma-12 (PC12) cells were divided into seven groups: Control group, Hypoxia group (Cells treated with CoCl 2 ), MALAT-1 group (Hypoxic cells treated with MALAT-1), NC group (Hypoxic cells treated with empty plasmid), MALAT-1 siRNA group (Hypoxic cells treated with siRNA MALAT-1), SB203580 group (Hypoxic cells treated with p38MAPK inhibitor), and MALAT-1 + SB20358 group. The content of reactive oxygen species (ROS), malondialdehyde (MDA), super oxide dismutase (SOD) and lactate dehydrogenase (LDH) was determined. Cell viability was detected by MTT assay. Apoptotic cells were observed by Hoechst 33258 and TUNEL staining assay. Mitochondrial membrane potential (MMP) was measured using JC1 vital dye. The decreased cell viability and increased expressions of MALAT-1 and p-p38 were observed in hypoxic PC12 cells time-dependently (P PC12 cells had an elevation in p-p38, ROS, MDA and LDH with the increased apoptotic cells, but a reduction in SOD and MMP, and these similar changes were more obvious in those hypoxic cells treated with MALAT-1 when compared with Controls (all P PC12 cells treated with SB203580 and MALAT-1 siRNA led to opposite results compared with MALAT-1 group (all P PC12 cells. MALAT-1 can promote the apoptosis and oxidative stress of PC12 cells by activating p38MAPK pathway, thus aggravating the damage of PC12 cells induced by chemical hypoxia. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling.

    Science.gov (United States)

    Hartwig, Kerstin; Fackler, Viktoria; Jaksch-Bogensperger, Heidi; Winter, Stefan; Furtner, Tanja; Couillard-Despres, Sebastien; Meier, Dieter; Moessler, Herbert; Aigner, Ludwig

    2014-11-01

    Cerebrolysin (EVER Neuro Pharma GmbH, Austria) is a peptidergic drug indicated for clinical use in stroke, traumatic brain injury and dementia. The therapeutic effect of Cerebrolysin is thought to ensure from its neurotrophic activity, which shares some properties with naturally occurring neurotrophic factors. However, the exact mechanism of action of Cerebrolysin is yet to be fully deciphered. This study aimed to investigate the neuroprotective effect of Cerebrolysin in a widely used in vitro model of hypoxia-induced neuronal cytotoxicity, namely cobalt chloride (CoCl2)-treatment of PC12 cells. CoCl2-cytotoxicity was indicated by a reduced cell-diameter, cell shrinkage, increased pro-apoptotic Caspase-activities and a decreased metabolic activity. Cerebrolysin maintained the cell-diameter of CoCl2-treated naïve PC12 cells, decreased the activation of Caspase 3/7 in CoCl2-stressed naïve PC12 cells and restored the cells' metabolic activity in CoCl2-impaired naïve and differentiated PC12 cells. Cerebrolysin treatment also decreased the levels of superoxide observed after exposure to CoCl2. Investigating the mechanism of action, we could demonstrate that Cerebrolysin application to CoCl2-stressed PC12 cells increased the phosphorylation of GSK3β, resulting in the inhibition of GSK3β. This might become clinically relevant for Alzheimer's disease, since GSK3β activity has been linked to the production of amyloid beta. Taken together, Cerebrolysin was found to have neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

  14. Multiparametric imaging of patient and tumour heterogeneity in non-small-cell lung cancer: quantification of tumour hypoxia, metabolism and perfusion

    Energy Technology Data Exchange (ETDEWEB)

    Elmpt, Wouter van; Zegers, Catharina M.L.; Reymen, Bart; Even, Aniek J.G.; Oellers, Michel; Troost, Esther G.C.; Lambin, Philippe [Maastricht University Medical Centre, Department of Radiation Oncology (MAASTRO), GROW - School for Oncology and Developmental Biology, Maastricht (Netherlands); Dingemans, Anne-Marie C. [Maastricht University Medical Centre, Department of Pulmonology, GROW - School for Oncology and Developmental Biology, Maastricht (Netherlands); Wildberger, Joachim E.; Das, Marco [Maastricht University Medical Centre, Department of Radiology, GROW - School for Oncology and Developmental Biology, Maastricht (Netherlands); Mottaghy, Felix M. [Maastricht University Medical Centre, Department of Nuclear Medicine, GROW - School for Oncology and Developmental Biology, Maastricht (Netherlands); University Hospital RWTH Aachen University, Department of Nuclear Medicine, Aachen (Germany)

    2016-02-15

    Multiple imaging techniques are nowadays available for clinical in-vivo visualization of tumour biology. FDG PET/CT identifies increased tumour metabolism, hypoxia PET visualizes tumour oxygenation and dynamic contrast-enhanced (DCE) CT characterizes vasculature and morphology. We explored the relationships among these biological features in patients with non-small-cell lung cancer (NSCLC) at both the patient level and the tumour subvolume level. A group of 14 NSCLC patients from two ongoing clinical trials (NCT01024829 and NCT01210378) were scanned using FDG PET/CT, HX4 PET/CT and DCE CT prior to chemoradiotherapy. Standardized uptake values (SUV) in the primary tumour were calculated for the FDG and hypoxia HX4 PET/CT scans. For hypoxia imaging, the hypoxic volume, fraction and tumour-to-blood ratio (TBR) were also defined. Blood flow and blood volume were obtained from DCE CT imaging. A tumour subvolume analysis was used to quantify the spatial overlap between subvolumes. At the patient level, negative correlations were observed between blood flow and the hypoxia parameters (TBR >1.2): hypoxic volume (-0.65, p = 0.014), hypoxic fraction (-0.60, p = 0.025) and TBR (-0.56, p = 0.042). At the tumour subvolume level, hypoxic and metabolically active subvolumes showed an overlap of 53 ± 36 %. Overlap between hypoxic sub-volumes and those with high blood flow and blood volume was smaller: 15 ± 17 % and 28 ± 28 %, respectively. Half of the patients showed a spatial mismatch (overlap <5 %) between increased blood flow and hypoxia. The biological imaging features defined in NSCLC tumours showed large interpatient and intratumour variability. There was overlap between hypoxic and metabolically active subvolumes in the majority of tumours, there was spatial mismatch between regions with high blood flow and those with increased hypoxia. (orig.)

  15. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

    Science.gov (United States)

    Favaro, Elena; Ramachandran, Anassuya; McCormick, Robert; Gee, Harriet; Blancher, Christine; Crosby, Meredith; Devlin, Cecilia; Blick, Christopher; Buffa, Francesca; Li, Ji-Liang; Vojnovic, Borivoj; Pires das Neves, Ricardo; Glazer, Peter; Iborra, Francisco; Ivan, Mircea; Ragoussis, Jiannis; Harris, Adrian L

    2010-04-26

    Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

  16. Hypoxia-inducible factors: coupling glucose metabolism and redox regulation with induction of the breast cancer stem cell phenotype.

    Science.gov (United States)

    Semenza, Gregg L

    2017-02-01

    Reduced oxygen availability (hypoxia) leads to increased production of reactive oxygen species (ROS) by the electron transport chain. Here, I review recent work delineating mechanisms by which hypoxia-inducible factor 1 (HIF-1) mediates adaptive metabolic responses to hypoxia, including increased flux through the glycolytic pathway and decreased flux through the tricarboxylic acid cycle, in order to decrease mitochondrial ROS production. HIF-1 also mediates increased flux through the serine synthesis pathway and mitochondrial one-carbon (folate cycle) metabolism to increase mitochondrial antioxidant production (NADPH and glutathione). Dynamic maintenance of ROS homeostasis is required for induction of the breast cancer stem cell phenotype in response to hypoxia or cytotoxic chemotherapy. Consistently, inhibition of phosphoglycerate dehydrogenase, the first enzyme of the serine synthesis pathway, in breast cancer cells impairs tumor initiation, metastasis, and response to cytotoxic chemotherapy. I discuss how these findings have important implications for understanding the logic of the tumor microenvironment and for improving therapeutic responses in women with breast cancer. © 2016 The Author.

  17. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

    Directory of Open Access Journals (Sweden)

    Elena Favaro

    2010-04-01

    Full Text Available Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1 and a microRNA, hsa-miR-210 (miR-210 which is associated with a poor prognosis.In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

  18. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    International Nuclear Information System (INIS)

    Doi, Nobutaka; Ogawa, Ryohei; Cui, Zheng-Guo; Morii, Akihiro; Watanabe, Akihiko; Kanayama, Shinji; Yoneda, Yuko; Kondo, Takashi

    2015-01-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl 2 confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype

  19. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Nobutaka [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Cui, Zheng-Guo [Department of Public Health, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Morii, Akihiro; Watanabe, Akihiko [Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Kanayama, Shinji; Yoneda, Yuko [New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan)

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  20. Reciprocal Regulation of Hypoxia-Inducible Factor 2α and GLI1 Expression Associated With the Radioresistance of Renal Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jiancheng [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China); Department of Urology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Wu, Kaijie [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China); Gao, Dexuan [Department of Urology, Shandong Provincial Hospital affiliated with Shandong University, Ji' nan (China); Zhu, Guodong; Wu, Dapeng; Wang, Xinyang; Chen, Yule; Du, Yuefeng; Song, Wenbin; Ma, Zhenkun [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China); Authement, Craig; Saha, Debabrata [Department of Urology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Hsieh, Jer-Tsong, E-mail: jt.hsieh@utsouthwestern.edu [Department of Urology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); He, Dalin, E-mail: dalinhe@yahoo.com [Department of Urology, First Affiliated Hospital of Medical School, Xi' an Jiaotong University, Xi' an (China)

    2014-11-15

    Purpose: Renal cell carcinoma (RCC) is often considered a radioresistant tumor, but the molecular mechanism underlying its radioresistance is poorly understood. This study explored the roles of hypoxia-inducible factor 2α (HIF2α) and sonic hedgehog (SHH)-GLI1 signaling in mediating the radioresistance of RCC cells and to unveil the interaction between these 2 signaling pathways. Methods and Materials: The activities of SHH-GLI1 signaling pathway under normoxia and hypoxia in RCC cells were examined by real-time polymerase chain reaction, Western blot, and luciferase reporter assay. The expression of HIF2α and GLI1 in RCC patients was examined by immunohistochemistry, and their correlation was analyzed. Furthermore, RCC cells were treated with HIF2α-specific shRNA (sh-HIF2α), GLI1 inhibitor GANT61, or a combination to determine the effect of ionizing radiation (IR) on RCC cells based on clonogenic assay and double-strand break repair assay. Results: RCC cells exhibited elevated SHH-GLI1 activities under hypoxia, which was mediated by HIF2α. Hypoxia induced GLI1 activation through SMO-independent pathways that could be ablated by PI3K inhibitor or MEK inhibitor. Remarkably, the SHH-GLI1 pathway also upregulated HIF2α expression in normoxia. Apparently, there was a positive correlation between HIF2α and GLI1 expression in RCC patients. The combination of sh-HIF2α and GLI1 inhibitor significantly sensitized RCC cells to IR. Conclusions: Cross-talk between the HIF2α and SHH-GLI1 pathways was demonstrated in RCC. Cotargeting these 2 pathways, significantly sensitizing RCC cells to IR, provides a novel strategy for RCC treatment.

  1. Hypoxia Inducible Factors have distinct and stage-specific roles during reprogramming of human cells to pluripotency

    Science.gov (United States)

    Mathieu, Julie; Zhou, Wenyu; Xing, Yalan; Sperber, Henrik; Ferreccio, Amy; Agoston, Zsuzsa; Kuppusamy, Kavitha T; Moon, Randall T; Ruohola-Baker, Hannele

    2014-01-01

    SUMMARY Pluripotent stem cells have distinct metabolic requirements, and reprogramming cells to pluripotency requires a shift from oxidative to glycolytic metabolism. Here, we show that this shift occurs early during reprogramming of human cells and requires Hypoxia Inducible Factors in a stage-specific manner. HIF1α and HIF2α are both necessary to initiate this metabolic switch and for acquisition of pluripotency, and stabilization of either protein during early phases of reprogramming is sufficient to induce the switch to glycolytic metabolism. In contrast, stabilization of HIF2α during later stages represses reprogramming, due at least in part to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL inhibits iPSC generation by repressing apoptotic caspase 3 activity specifically in cells undergoing reprogramming, but not hESCs, and inhibiting TRAIL activity enhances hiPSC generation. These results shed light on the mechanisms underlying the metabolic shifts associated with acquisition of a pluripotent identity during reprogramming. PMID:24656769

  2. mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells.

    Science.gov (United States)

    Kuo, Chung-Wen; Tsai, Meng-Han; Lin, Tsu-Kung; Tiao, Mao-Meng; Wang, Pei-Wen; Chuang, Jiin-Haur; Chen, Shang-Der; Liou, Chia-Wei

    2017-06-07

    Mitochondria consume O₂ to produce ATP and are critical for adaption of hypoxia, but the role of mitochondria in HIF-1α pathway is as yet unclear. In this study, mitochondrial DNA (mtDNA) enriched (SK-N-AS) and depleted (ρ⁰) cells of neuroblastoma were cultured in a hypoxic chamber to simulate a hypoxic condition and then the major components involved in mitochondrial related pathways, hypoxia-inducible factor 1α (HIF-1α) and reactive oxygen species (ROS) were measured. The results showed that hypoxia-stimulated exposure elevated expression of HIF-1α, which was additionally influenced by level of generated ROS within the cytosol. Moreover, elevation of HIF-1α also resulted in increases of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase kinase 1 (PDK1) in both hypoxic cells. The expression of mitochondrial biogenesis related proteins and metabolic components were noted to increase significantly in hypoxic SK-N-AS cells, indicating that mtDNA was involved in mitochondrial retrograde signaling and metabolic pathways. An analysis of dynamic proteins found elevated levels of HIF-1α causing an increased expression of dynamin-related protein 1 (DRP1) during hypoxia; further, the existence of mtDNA also resulted in higher expression of DRP1 during hypoxia. By using siRNA of HIF-1α or DRP1, expression of DRP1 decreased after suppression of HIF-1α; moreover, the expression of HIF-1α was also affected by the suppression of DRP1. In this study, we demonstrated that mtDNA is a mediator of HIF-1α in eliciting metabolic reprogramming, and mitochondrial biogenesis. Identification of a mutual relationship between HIF-1α and DRP1 may be a critical tool in the future development of clinical applications.

  3. Effects of copper sulfate-oxidized or myeloperoxidase- modified LDL on lipid loading and programmed cell death in macrophages under hypoxia

    Directory of Open Access Journals (Sweden)

    Vlaminck B

    2014-09-01

    Full Text Available Benoit Vlaminck,1 Damien Calay,1 Marie Genin,1 Aude Sauvage,1 Noelle Ninane,1 Karim Zouaoui Boudjeltia,2 Martine Raes,1 Carine Michiels1 1Laboratory of Biochemistry and Cellular Biology (URBC, Namur Research Institute for Life Sciences (NARILIS, University of Namur, Namur, Belgium; 2Laboratory of Experimental Medicine (ULB 222 Unit, Universite Libre de Bruxelles, CHU de Charleroi, Charleroi, Belgium Abstract: Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia. Keywords: Ox-LDL, myeloperoxidase, hypoxia, UPR, apoptosis, autophagy, macrophages

  4. Different response to hypoxia of adipose-derived multipotent cells from obese subjects with and without metabolic syndrome.

    Directory of Open Access Journals (Sweden)

    Wilfredo Oliva-Olivera

    Full Text Available Multiple studies suggest that hypoxia, together with inflammation, could be one of the phenomena involved in the onset and progression of obesity-related insulin resistance. In addition, dysfunction of adipose tissue in obese subjects with metabolic syndrome is associated with decreased angiogenesis. However, some subjects with a high body mass index do not develop metabolic abnormalities associated with obesity. The aim of the current study was to examine the neovascular properties of visceral adipose tissue-derived multipotent mesenchymal cells subjected to hypoxia (hypox-visASCs from normal-weight subjects (Nw and obese patients with metabolic syndrome (MS and without metabolic syndrome (NonMS.This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. Eight patients who underwent either bariatric surgery or cholecystectomy (27 patients participated in the study. Visceral adipose tissue samples from Nw, MS and NonMS subjects were processed by enzymatic digestion. VisASCs cultured under hypoxic conditions were characterized by tubule formation assay, ELISA, flow cytometry, migration rate, and qRT-PCR, and the effects of visASCs-conditioned medium on survival and endothelial cell tubule formation were evaluated.Hypox-visASCs from NonMS subjects showed a greater capacity for tubule formation than hypox-visASCs from Nw and MS subjects. The lower percentage of CD140b+/CD44+ and CD140b+/CD184+ cells observed in hypox-visASCs from NonMS subjects compared to MS subjects was accompanied not only by a lower migration rate from the chemotactic effects of stromal cell derived factor 1α, but also by lower levels of NOX5 mRNA expression. While the levels of monocyte chemoattractant protein 1 mRNA expressed by hypox-visASCs correlated positively with the body mass index and waist circumference of the subjects, the concentration of vascular endothelial growth factor present in hypox-visASC-conditioned culture medium

  5. Different response to hypoxia of adipose-derived multipotent cells from obese subjects with and without metabolic syndrome

    Science.gov (United States)

    Moreno-Indias, Isabel; Coín-Aragüez, Leticia; Lhamyani, Said; Alcaide Torres, Juan; Fernández-Veledo, Sonia; Vendrell, Joan; Camargo, Antonio; El Bekay, Rajaa; Tinahones, Francisco José

    2017-01-01

    Background/Objectives Multiple studies suggest that hypoxia, together with inflammation, could be one of the phenomena involved in the onset and progression of obesity-related insulin resistance. In addition, dysfunction of adipose tissue in obese subjects with metabolic syndrome is associated with decreased angiogenesis. However, some subjects with a high body mass index do not develop metabolic abnormalities associated with obesity. The aim of the current study was to examine the neovascular properties of visceral adipose tissue-derived multipotent mesenchymal cells subjected to hypoxia (hypox-visASCs) from normal-weight subjects (Nw) and obese patients with metabolic syndrome (MS) and without metabolic syndrome (NonMS). Methods This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. Eight patients who underwent either bariatric surgery or cholecystectomy (27 patients) participated in the study. Visceral adipose tissue samples from Nw, MS and NonMS subjects were processed by enzymatic digestion. VisASCs cultured under hypoxic conditions were characterized by tubule formation assay, ELISA, flow cytometry, migration rate, and qRT-PCR, and the effects of visASCs-conditioned medium on survival and endothelial cell tubule formation were evaluated. Results Hypox-visASCs from NonMS subjects showed a greater capacity for tubule formation than hypox-visASCs from Nw and MS subjects. The lower percentage of CD140b+/CD44+ and CD140b+/CD184+ cells observed in hypox-visASCs from NonMS subjects compared to MS subjects was accompanied not only by a lower migration rate from the chemotactic effects of stromal cell derived factor 1α, but also by lower levels of NOX5 mRNA expression. While the levels of monocyte chemoattractant protein 1 mRNA expressed by hypox-visASCs correlated positively with the body mass index and waist circumference of the subjects, the concentration of vascular endothelial growth factor present in hypox

  6. The role of HIF-1 in up-regulating MICA expression on human renal proximal tubular epithelial cells during hypoxia/reoxygenation

    Directory of Open Access Journals (Sweden)

    Li Fu S

    2010-11-01

    Full Text Available Abstract Background Human major histocompatibility complex class I-related chain A (MICA plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1 is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity. Results We find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2 cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased. Conclusions These results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is

  7. Hypoxia-inducible factor-1α and Wnt/β-catenin signaling pathways promote the invasion of hypoxic gastric cancer cells.

    Science.gov (United States)

    Liu, Hong-Lan; Liu, Dang; Ding, Guang-Rong; Liao, Peng-Fei; Zhang, Jun-Wen

    2015-09-01

    The present study aimed to examine the association between hypoxia-inducible factor (HIF)-1α and the Wnt/β-catenin signaling pathway in a hypoxic environment. The study also aimed to explore the possible mechanisms underlying the invasion of hypoxic gastric cancer cells in vitro and in vivo. The pcDNA™ 6.2‑GW/EmGFP‑miR‑β‑catenin plasmid was transfected into SGC‑7901 gastric cancer cells, resulting in cells with stable suppression of β‑catenin expression. The biological characteristics of the control, liposome, negative control, β‑catenin knockdown, hypoxia and hypoxia β‑catenin knockdown groups were tested using an invasion assay. The differences in the invasive capacity of the control, negative control and liposome groups were not statistically significant. However, the hypoxia group demonstrated a significantly enhanced invasive capacity, as compared with that in the control group (Phypoxic and control cells was high alongside increased HIF‑1α, β‑catenin, uPA and MMP‑7 levels according to western blot and immunohistochemical analyses, while growth and protein levels of tumors from hypoxic β‑catenin knockdown cells were significantly lower and those of β‑catenin knockdown cells were lowest. In conclusion, these results suggested that HIF‑1α activation was able to regulate the Wnt/β‑catenin pathway, and that HIF‑1α may be controlled by the Wnt/β‑catenin pathway. A potential mechanism underlying SGC‑7901 tumorigenicity is the activation of the Wnt/β‑catenin signaling pathway, which activates uPA and MMP‑7 expression and contributes to the enhanced invasion of hypoxic cancer cells.

  8. Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins.

    Science.gov (United States)

    Gogate, Shilpa S; Nasser, Rena; Shapiro, Irving M; Risbud, Makarand V

    2011-07-01

    To determine whether hypoxia and hypoxia-inducible factor (HIF) proteins regulate expression of β-1,3-glucuronyltransferase 1 (GlcAT-1), a key enzyme in glycosaminoglycan synthesis in nucleus pulposus cells. Real-time reverse transcriptase-polymerase chain reaction and Western blotting were used to measure GlcAT-1 expression. Transfections were performed to determine the effect of HIF-1α and HIF-2α on GlcAT-1 promoter activity. Under hypoxic conditions there was an increase in GlcAT-1 expression; a significant increase in promoter activity was seen both in nucleus pulposus cells and in N1511 chondrocytes. We investigated whether HIF controlled GlcAT-1 expression. Suppression of HIF-1α and HIF-2α induced GlcAT-1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA-HIF-1α as well as with CA-HIF-2α suppressed GlcAT-1 promoter activity only in nucleus pulposus cells, suggesting a cell type-specific regulation. Site-directed mutagenesis and deletion constructs were used to further confirm the suppressive effect of HIFs on GlcAT-1 promoter function in nucleus pulposus cells. Although it was evident that interaction of HIF with hypoxia-responsive elements resulted in suppression of basal promoter activity, it was not necessary for transcriptional suppression. This result suggested both a direct and an indirect mode of regulation, possibly through recruitment of a HIF-dependent repressor. Finally, we showed that hypoxic expression of GlcAT-1 was also partially dependent on MAPK signaling. These studies demonstrate that hypoxia regulates GlcAT-1 expression through a signaling network comprising both activator and suppressor molecules, and that this regulation is unique to nucleus pulposus cells. Copyright © 2011 by the American College of Rheumatology.

  9. Hypoxia increases membrane metallo-endopeptidase expression in a novel lung cancer ex vivo model - role of tumor stroma cells

    NARCIS (Netherlands)

    K. Leithner (Katharina); C. Wohlkoenig (Christoph); E. Stacher (Elvira); J. Lindenmann (Jörg); N.A. Hofmann (Nicole); B. Gallé (Birgit); C. Guelly (Christian); F. Quehenberger (Franz); P. Stiegler (Philipp); F.-M. Smolle-Jüttner (Freyja-Maria); J.N.J. Philipsen (Sjaak); H.H. Popper (Helmut); A. Hrzenjak (Andelko); A. Olschewski (Andrea); H. Olschewski (Horst)

    2014-01-01

    textabstractBackground: Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied in vitro, however, they may differ in vivo due to the specific tumor microenvironment. In this study gene expression profiles were obtained from fresh human lung

  10. Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer- and metastasis-initiating cells

    Science.gov (United States)

    Mimeault, Murielle; Batra, Surinder K

    2013-01-01

    Accumulating lines of experimental evidence have revealed that hypoxia-inducible factors, HIF-1α and HIF-2α, are key regulators of the adaptation of cancer- and metastasis-initiating cells and their differentiated progenies to oxygen and nutrient deprivation during cancer progression under normoxic and hypoxic conditions. Particularly, the sustained stimulation of epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), stem cell factor (SCF) receptor KIT, transforming growth factor-β receptors (TGF-βRs) and Notch and their downstream signalling elements such as phosphatidylinositol 3′-kinase (PI3K)/Akt/molecular target of rapamycin (mTOR) may lead to an enhanced activity of HIFs. Moreover, the up-regulation of HIFs in cancer cells may also occur in the hypoxic intratumoral regions formed within primary and secondary neoplasms as well as in leukaemic cells and metastatic prostate and breast cancer cells homing in the hypoxic endosteal niche of bone marrow. The activated HIFs may induce the expression of numerous gene products such as induced pluripotency-associated transcription factors (Oct-3/4, Nanog and Sox-2), glycolysis- and epithelial-mesenchymal transition (EMT) programme-associated molecules, including CXC chemokine receptor 4 (CXCR4), snail and twist, microRNAs and angiogenic factors such as vascular endothelial growth factor (VEGF). These gene products in turn can play critical roles for high self-renewal ability, survival, altered energy metabolism, invasion and metastases of cancer cells, angiogenic switch and treatment resistance. Consequently, the targeting of HIF signalling network and altered metabolic pathways represents new promising strategies to eradicate the total mass of cancer cells and improve the efficacy of current therapies against aggressive and metastatic cancers and prevent disease relapse. PMID:23301832

  11. Cell-Based Phenotyping Reveals QTL for Membrane Potential Maintenance Associated with Hypoxia and Salinity Stress Tolerance in Barley

    Directory of Open Access Journals (Sweden)

    Muhammad B. Gill

    2017-11-01

    Full Text Available Waterlogging and salinity are two major abiotic stresses that hamper crop production world-wide resulting in multibillion losses. Plant abiotic stress tolerance is conferred by many interrelated mechanisms. Amongst these, the cell’s ability to maintain membrane potential (MP is considered to be amongst the most crucial traits, a positive relationship between the ability of plants to maintain highly negative MP and its tolerance to both salinity and waterlogging stress. However, no attempts have been made to identify quantitative trait loci (QTL conferring this trait. In this study, the microelectrode MIFE technique was used to measure the plasma membrane potential of epidermal root cells of 150 double haploid (DH lines of barley (Hordeum vulgare L. from a cross between a Chinese landrace TX9425 and Japanese malting cultivar Naso Nijo under hypoxic conditions. A major QTL for the MP in the epidermal root cells in hypoxia-exposed plants was identified. This QTL was located on 2H, at a similar position to the QTL for waterlogging and salinity tolerance reported in previous studies. Further analysis confirmed that MP showed a significant contribution to both waterlogging and salinity tolerance. The fact that the QTL for MP was controlled by a single major QTL illustrates the power of the single-cell phenotyping approach and opens prospects for fine mapping this QTL and thus being more effective in marker assisted selection.

  12. Hypoxic Culture Promotes Dopaminergic-Neuronal Differentiation of Nasal Olfactory Mucosa Mesenchymal Stem Cells via Upregulation of Hypoxia-Inducible Factor-1α.

    Science.gov (United States)

    Zhuo, Yi; Wang, Lei; Ge, Lite; Li, Xuan; Duan, Da; Teng, Xiaohua; Jiang, Miao; Liu, Kai; Yuan, Ting; Wu, Pei; Wang, Hao; Deng, Yujia; Xie, Huali; Chen, Ping; Xia, Ying; Lu, Ming

    2017-08-01

    Olfactory mucosa mesenchymal stem cells (OM-MSCs) display significant clonogenic activity and may be easily propagated for Parkinson's disease therapies. Methods of inducing OM-MSCs to differentiate into dopaminergic (DAergic) neurons using olfactory ensheathing cells (OECs) are thus an attractive topic of research. We designed a hypoxic induction protocol to generate DAergic neurons from OM-MSCs using a physiological oxygen (O 2 ) level of 3% and OEC-conditioned medium (OCM; HI group). The normal induction (NI) group was cultured in O 2 at ambient air level (21%). The role of hypoxia-inducible factor-1α (HIF-1α) in the differentiation of OM-MSCs under hypoxia was investigated by treating cells with an HIF-1α inhibitor before induction (HIR group). The proportions of β-tubulin- and tyrosine hydroxylase (TH)-positive cells were significantly increased in the HI group compared with the NI and HIR groups, as shown by immunocytochemistry and Western blotting. Furthermore, the level of dopamine was significantly increased in the HI group. A slow outward potassium current was recorded in differentiated cells after 21 d of induction using whole-cell voltage-clamp tests. A hypoxic environment thus promotes OM-MSCs to differentiate into DAergic neurons by increasing the expression of HIF-1α and by activating downstream target gene TH. This study indicated that OCM under hypoxic conditions could significantly upregulate key transcriptional factors involved in the development of DAergic neurons from OM-MSCs, mediated by HIF-1α. Hypoxia promotes DAergic neuronal differentiation of OM-MSCs, and HIF-1α may play an important role in hypoxia-inducible pathways during DAergic lineage specification and differentiation in vitro.

  13. Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1

    Directory of Open Access Journals (Sweden)

    Federica Raggi

    2017-09-01

    Full Text Available Macrophages (Mf are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1 and alternatively activated (anti-inflammatory M2 subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.

  14. HPV status, cancer stem cell marker expression, hypoxia gene signatures and tumour volume identify good prognosis subgroups in patients with HNSCC after primary radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG)

    DEFF Research Database (Denmark)

    Linge, Annett; Lohaus, Fabian; Löck, Steffen

    2016-01-01

    OBJECTIVE: To investigate the impact of the tumour volume, HPV status, cancer stem cell (CSC) marker expression and hypoxia gene signatures, as potential markers of radiobiological mechanisms of radioresistance, in a contemporary cohort of patients with locally advanced head and neck squamous cell...... expression on LRC. The tumour hypoxia classification showed a significant impact only for small tumours. In multivariate analyses an independent correlation of tumour volume, SLC3A2 expression, and the 15-gene hypoxia signature with LRC was identified (CD44 protein n/a because of no event in the CD44...

  15. Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges

    Directory of Open Access Journals (Sweden)

    Tangni Gómez-Leduc

    2017-09-01

    Full Text Available Umbilical cord blood (UCB is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2 and transforming growth factor-β (TGF-β1 and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O2 or hypoxia (<5% O2 for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-β1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13 expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering.

  16. Effects of hypoxia condition in embryogenic callus growth of soybean cell culture

    Science.gov (United States)

    Damanik, R. I.; Manurung, B. H.; Bayu, E. S.

    2018-02-01

    The study was performed at Tissue Culture Laboratory, Agrotechnology Department, University of Sumatera Utara, to investigate the effect of plant growth regulator (PGR) and embryogenic callus performance soybean cultivars on hypoxia condition. This research had two stages, induction of embryogenic callus and analysis metabolism of callus after hypoxic condition with T-test. The analysis was used factorial Completely Randomized Design with two factors. The first factors were cultivars of soybean (Baluran, Gepak Kuning, and Grobogan) and the second factors were combinations of PGR (5 mg/l 2,4-D + 1 mg/l BAP, 10 mg/l 2,4-D + 1.5 mg/l BAP, and 15 mg/l 2,4-D + 2 mg/l BAP). The result showed the cultivars, combination of PGR, and interaction between cultivars and PGR gave significant effect to weight callus. The result of T-test showed that in hypoxic condition, POD enzyme exercise on Gepak Kuning’s callus in 5 mg/l 2,4-D + 1 mg/l BAP was different before and after hypoxic condition.

  17. Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM

    International Nuclear Information System (INIS)

    Burgman, Paul; O'Donoghue, Joseph A.; Lewis, Jason S.; Welch, Michael J.; Humm, John L.; Ling, C. Clifton

    2005-01-01

    Background: Cu-diacetyl-bis(N 4 -methylthiosemicarbazone) [Cu-ATSM] is a potential marker for tumor hypoxia that has been under evaluation for clinical use. In this study, we examined the mechanisms underlying the uptake of 64 Cu in cells incubated with 64 Cu-ATSM. Methods: The in vitro uptake of 64 Cu was determined as a function of oxygenation conditions and incubation time with 64 Cu-ATSM using four and two tumor cell lines of human origin and rodent origin, respectively. Additionally, the rate of 64 Cu efflux and Cu-ATSM metabolism was determined. Results: 64 Cu accumulation is rapid during the first 0.5-1 h of incubation. It is highest in anoxic cells but is also significant in normoxic cells. After this initial period, the level of intracellular 64 Cu varies depending on the cell line and the oxygenation conditions and, in some circumstances, may decrease. During the first 0.5-1 h, the ratio of 64 Cu levels between anoxic and normoxic cells is ∼2:10 and that between hypoxic (0.5% O 2 ) and normoxic cells is ∼1:2.5, depending on the cell line. These ratios generally decrease at longer times. The 64 Cu-ATSM compound was found to be metabolized during incubation in a manner dependent on oxygenation conditions. Within 2 h under anoxic conditions, 64 Cu-ATSM could no longer be detected, although 60-90% of the amount of 64 Cu added as 64 Cu-ATSM was present in the medium. Non-ATSM 64 Cu was taken up by the cells, albeit at a much slower rate. Efflux rates of 64 Cu were found to be cell line dependent and appeared to be inversely correlated with the final 64 Cu uptake levels under anoxic conditions. Conclusion: The uptake and retention of 64 Cu and their relation to oxygenation conditions were found to be cell line dependent. Given the complexities in the oxygen dependence and cell line-dependent kinetics of uptake and retention of Cu following exposure to Cu-ATSM, the clinical utility of this compound may be disease site specific

  18. The regulation of transcriptional repression in hypoxia

    OpenAIRE

    Cavadas, Miguel A.S.; Cheong, Alex; Taylor, Cormac T.

    2017-01-01

    A sufficient supply molecular oxygen is essential for the maintenance of physiologic metabolism and bioenergetic homeostasis for most metazoans. For this reason, mechanisms have evolved for eukaryotic cells to adapt to conditions where oxygen demand exceeds supply (hypoxia). These mechanisms rely on the modification of pre-existing proteins, translational arrest and transcriptional changes. The hypoxia inducible factor (HIF; a master regulator of gene induction in response to hypoxia) is resp...

  19. Inflammation and hypoxia in the kidney: friends or foes?

    Science.gov (United States)

    Haase, Volker H

    2015-08-01

    Hypoxic injury is commonly associated with inflammatory-cell infiltration, and inflammation frequently leads to the activation of cellular hypoxia response pathways. The molecular mechanisms underlying this cross-talk during kidney injury are incompletely understood. Yamaguchi and colleagues identify CCAAT/enhancer-binding protein δ as a cytokine- and hypoxia-regulated transcription factor that fine-tunes hypoxia-inducible factor-1 signaling in renal epithelial cells and thus provide a novel molecular link between hypoxia and inflammation in kidney injury.

  20. Sirtuins 1-7 expression in human adipose-derived stem cells from subcutaneous and visceral fat depots: influence of obesity and hypoxia.

    Science.gov (United States)

    Mariani, Stefania; Di Rocco, Giuliana; Toietta, Gabriele; Russo, Matteo A; Petrangeli, Elisa; Salvatori, Luisa

    2017-09-01

    The sirtuin family comprises seven NAD + -dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins' levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins' expression and clinical and molecular parameters were also analyzed. We found that sirtuin1-6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1-6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins' expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins' levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral

  1. Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha

    International Nuclear Information System (INIS)

    McFarlane, Steven; Nicholl, Mary Jane; Sutherland, Jane S.; Preston, Chris M.

    2011-01-01

    The cellular protein hypoxia-inducible factor 1 alpha (HIF-1α) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1α was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1α-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1α to occur. HIF-1α controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology.

  2. Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers.

    Science.gov (United States)

    He, Fuqiu; Deng, Xuelong; Wen, Bixiu; Liu, Yueping; Sun, Xiaorong; Xing, Ligang; Minami, Akiko; Huang, Yunhong; Chen, Qing; Zanzonico, Pat B; Ling, C Clifton; Li, Gloria C

    2008-10-15

    Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer (18)F-FMISO and the reporter substrate (124)I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: (124)I-FIAU, (18)F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of (124)I-FIAU and (18)F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

  3. Micro Regional Heterogeneity of 64Cu-ATSM and 18F-FDG Uptake in Canine Soft Tissue Sarcomas: Relation to Cell Proliferation, Hypoxia and Glycolysis.

    Science.gov (United States)

    Zornhagen, Kamilla Westarp; Hansen, Anders E; Oxboel, Jytte; Clemmensen, Andreas E; El Ali, Henrik H; Kristensen, Annemarie T; Kjær, Andreas

    2015-01-01

    Tumour microenvironment heterogeneity is believed to play a key role in cancer progression and therapy resistance. However, little is known about micro regional distribution of hypoxia, glycolysis and proliferation in spontaneous solid tumours. The overall aim was simultaneous investigation of micro regional heterogeneity of 64Cu-ATSM (hypoxia) and 18F-FDG (glycolysis) uptake and correlation to endogenous markers of hypoxia, glycolysis, proliferation and angiogenesis to better therapeutically target aggressive tumour regions and prognosticate outcome. Exploiting the different half-lives of 64Cu-ATSM (13 h) and 18F-FDG (2 h) enabled simultaneous investigation of micro regional distribution of hypoxia and glycolysis in 145 tumour pieces from four spontaneous canine soft tissue sarcomas. Pairwise measurements of radioactivity and gene expression of endogenous markers of hypoxia (HIF-1α, CAIX), glycolysis (HK2, GLUT1 and GLUT3), proliferation (Ki-67) and angiogenesis (VEGFA and TF) were performed. Dual tracer autoradiography was compared with Ki-67 immunohistochemistry. Micro regional heterogeneity in hypoxia and glycolysis within and between tumour sections of each tumour piece was observed. The spatial distribution of 64Cu-ATSM and 18F-FDG was rather similar within each tumour section as reflected in moderate positive significant correlations between the two tracers (ρ = 0.3920-0.7807; p = 0.0180 -pixel-to-pixel comparisons of autoradiographies and gamma counting of tumour pieces. 64Cu-ATSM and 18F-FDG correlated positively with gene expression of GLUT1 and GLUT3, but negatively with HIF-1α and CAIX. Significant positive correlations were seen between Ki-67 gene expression and 64Cu-ATSM (ρ = 0.5578, p = 0.0004) and 18F-FDG (ρ = 0.4629-0.7001, p = 0.0001-0.0151). Ki-67 gene expression more consistently correlated with 18F-FDG than with 64Cu-ATSM. Micro regional heterogeneity of hypoxia and glycolysis was documented in spontaneous canine soft tissue sarcomas. 64Cu

  4. Micro Regional Heterogeneity of 64Cu-ATSM and 18F-FDG Uptake in Canine Soft Tissue Sarcomas: Relation to Cell Proliferation, Hypoxia and Glycolysis.

    Directory of Open Access Journals (Sweden)

    Kamilla Westarp Zornhagen

    Full Text Available Tumour microenvironment heterogeneity is believed to play a key role in cancer progression and therapy resistance. However, little is known about micro regional distribution of hypoxia, glycolysis and proliferation in spontaneous solid tumours. The overall aim was simultaneous investigation of micro regional heterogeneity of 64Cu-ATSM (hypoxia and 18F-FDG (glycolysis uptake and correlation to endogenous markers of hypoxia, glycolysis, proliferation and angiogenesis to better therapeutically target aggressive tumour regions and prognosticate outcome.Exploiting the different half-lives of 64Cu-ATSM (13 h and 18F-FDG (2 h enabled simultaneous investigation of micro regional distribution of hypoxia and glycolysis in 145 tumour pieces from four spontaneous canine soft tissue sarcomas. Pairwise measurements of radioactivity and gene expression of endogenous markers of hypoxia (HIF-1α, CAIX, glycolysis (HK2, GLUT1 and GLUT3, proliferation (Ki-67 and angiogenesis (VEGFA and TF were performed. Dual tracer autoradiography was compared with Ki-67 immunohistochemistry.Micro regional heterogeneity in hypoxia and glycolysis within and between tumour sections of each tumour piece was observed. The spatial distribution of 64Cu-ATSM and 18F-FDG was rather similar within each tumour section as reflected in moderate positive significant correlations between the two tracers (ρ = 0.3920-0.7807; p = 0.0180 -<0.0001 based on pixel-to-pixel comparisons of autoradiographies and gamma counting of tumour pieces. 64Cu-ATSM and 18F-FDG correlated positively with gene expression of GLUT1 and GLUT3, but negatively with HIF-1α and CAIX. Significant positive correlations were seen between Ki-67 gene expression and 64Cu-ATSM (ρ = 0.5578, p = 0.0004 and 18F-FDG (ρ = 0.4629-0.7001, p = 0.0001-0.0151. Ki-67 gene expression more consistently correlated with 18F-FDG than with 64Cu-ATSM.Micro regional heterogeneity of hypoxia and glycolysis was documented in spontaneous canine

  5. Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, Justin; Holmuhamedov, Ekhson; Zhang, Xun; Lovelace, Gregory L.; Smith, Charles D. [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC (United States); Lemasters, John J., E-mail: JJLemasters@musc.edu [Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC (United States); Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC (United States)

    2013-11-15

    Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50 μM of each tetracycline-derived compound 20 min prior to exposure to 500 μM iodoacetic acid plus 1 mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs–Ringer–HEPES buffer at pH 6.2 for 4 h prior to reoxygenation at pH 7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca{sup 2+} uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca{sup 2+} uptake and suppressed the Ca{sup 2+}-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca{sup 2+} uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU. - Highlights: • Minocycline and doxycycline are the only cytoprotective tetracyclines of those tested • Cytoprotective tetracyclines inhibit the MPT and mitochondrial calcium and iron uptake. • Cytoprotective

  6. Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

    Directory of Open Access Journals (Sweden)

    Catherine To

    Full Text Available Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs, but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

  7. Bifurcate effects of glucose on caspase-independent cell death during hypoxia

    International Nuclear Information System (INIS)

    Aki, Toshihiko; Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi

    2010-01-01

    We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

  8. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Yonghui; Kato, Tomohisa; Furu, Moritoshi [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Nasu, Akira [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University (Japan); Kajita, Yoichiro [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Urology, Graduate School of Medicine, Kyoto University (Japan); Mitsui, Hiroto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Musculoskeletal Medicine, Graduate School of Medical Sciences, Nagoya City University (Japan); Ueda, Michiko [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Aoyama, Tomoki [Human Health Sciences, Graduate School of Medicine, Kyoto University (Japan); Nakayama, Tomitaka; Nakamura, Takashi [Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University (Japan); Center for iPS Cell Research and Application, Institute for Integrated Cell - Material Sciences, Kyoto University (Japan)

    2010-01-15

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO{sub 2}) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO{sub 2}) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  9. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    International Nuclear Information System (INIS)

    Jin, Yonghui; Kato, Tomohisa; Furu, Moritoshi; Nasu, Akira; Kajita, Yoichiro; Mitsui, Hiroto; Ueda, Michiko; Aoyama, Tomoki; Nakayama, Tomitaka; Nakamura, Takashi; Toguchida, Junya

    2010-01-01

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO 2 ) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO 2 ) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  10. [Birth hypoxia].

    Science.gov (United States)

    Větr, M

    2015-03-01

    Evaluation of the commonly used laboratory and clinical parameters of the newborn shortly after birth. Check thresholds acidemia, and in relation to the method of termination of pregnancy. Retrospective epidemiological study. Department of Obstetrics and Gynecology, University Hospital, Olomouc. Of the 26,869 children born in the years 2000 to 2013 Inclusion criteria (complete clinical and laboratory findings after birth) fulfill 23,471 (87.4%) neonates. Methods for evaluation of newborns included Apgar score calculation and arterial umbilical cord blood pH and lactate analysis. A total of 0.7% (157) of the neonates had severe acidosis pH below 7.00 arterial umbilical cord blood, its prevalence varies annually between 0.1 to 1.1%. Cutoff lactate in relation to pH reserves. Operating cesarean births in particular accounts for more than half of those with worse clinical findings Apgar and pH <7.00, but only 30% supratreshold lactate values. Also worse clinical evaluation after caesarean section is not in accordance with the laboratory findings. Vaginal surgery, especially forceps have a significant share of severe acidosis than cesarean, regardless of their frequency. Risk factor of forceps to pH less 7.00,OR = 9.28 (5.39 -15.77), P = 0.0000000, while caesarean to pH less 7,00 had OR = 1.52 (1.08 to 2.14), P = 0.01408156. The results obtained confirm that acidosis after birth is quite common, although they may not have response on the clinical condition of the newborn after birth. Evaluation of Apgar is little objective for the detection of hypoxia during birth and is influenced by the immaturity of newborn and method of delivery. Lactate levels may contribute to an objective assessment of hypoxia during birth. Values above 6.3 mmol/l can be considered an important indicator of newborn acidosis and birth hypoxia.

  11. Conversion of Stationary to Invasive Tumor Initiating Cells (TICs): Role of Hypoxia in Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) Trafficking

    Science.gov (United States)

    Li, Jian; Zucker, Stanley; Pulkoski-Gross, Ashleigh; Kuscu, Cem; Karaayvaz, Mihriban; Ju, Jingfang; Yao, Herui; Song, Erwei; Cao, Jian

    2012-01-01

    Emerging evidence has implicated the role of tumor initiating cells (TICs) in the process of cancer metastasis. The mechanism underlying the conversion of TICs from stationary to invasive remains to be characterized. In this report, we employed less invasive breast cancer TICs, SK-3rd, that displays CD44high/CD24low with high mammosphere-forming and tumorigenic capacities, to investigate the mechanism by which stationary TICs are converted to invasive TICs. Invasive ability of SK-3rd TICs was markedly enhanced when the cells were cultured under hypoxic conditions. Given the role of membrane type 1-matrix metalloproteinase (MT1-MMP) in cancer invasion/metastasis, we explored a possible involvement of MT1-MMP in hypoxia-induced TIC invasion. Silencing of MT1-MMP by a shRNA approach resulted in diminution of hypoxia-induced cell invasion in vitro and metastasis in vivo. Under hypoxic conditions, MT1-MMP redistributed from cytoplasmic storage pools to the cell surface of TICs, which coincides with the increased cell invasion. In addition, CD44, a cancer stem-like cell marker, inversely correlated with increased cell surface MT1-MMP. Interestingly, cell surface MT1-MMP gradually disappeared when the hypoxia-treated cells were switched to normoxia, suggesting the plasticity of TICs in response to oxygen content. Furthermore, we dissected the pathways leading to upregulated MT1-MMP in cytoplasmic storage pools under normoxic conditions, by demonstrating a cascade involving Twist1-miR10b-HoxD10 leading to enhanced MT1-MMP expression in SK-3rd TICs. These observations suggest that MT1-MMP is a key molecule capable of executing conversion of stationary TICs to invasive TICs under hypoxic conditions and thereby controlling metastasis. PMID:22679501

  12. Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

    Directory of Open Access Journals (Sweden)

    Bernhard F Gibbs

    Full Text Available Stem cell factor (SCF is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1 in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

  13. Hypoxia-induced aggressiveness of pancreatic cancer cells is due to increased expression of VEGF, IL-6 and miR-21, which can be attenuated by CDF treatment.

    Directory of Open Access Journals (Sweden)

    Bin Bao

    Full Text Available Hypoxia is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia mediated over-expression of hypoxia-inducible factor (HIF has been shown to be associated with therapeutic resistance, and contributes to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT, maintenance of cancer stem cell (CSC functions, and also maintains the vicious cycle of inflammation-all which lead to therapeutic resistance. However, the precise molecular mechanism(s by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC signature genes Nanog, Oct4 and EZH2 consistent with increased cell migration/invasion and angiogenesis, and the formation of pancreatospheres, concomitant with increased expression of miR-21 and miR-210 in human pancreatic cancer (PC cells. The treatment of PC cells with CDF, a novel synthetic compound inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxia. CDF also led to decreased cell migration/invasion, angiogenesis, and formation of pancreatospheres under hypoxia. Moreover, CDF decreased gene expression of miR-21, miR-210, IL-6, HIF-1α, VEGF, and CSC signatures in vivo in a mouse orthotopic model of human PC. Collectively, these results suggest that the anti-tumor activity of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.

  14. Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

    DEFF Research Database (Denmark)

    Bath, Chris; Yang, Sufang; Muttuvelu, Danson

    2013-01-01

    The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth ...

  15. The l1-l2 regularization framework unmasks the hypoxia signature hidden in the transcriptome of a set of heterogeneous neuroblastoma cell lines.

    Science.gov (United States)

    Fardin, Paolo; Barla, Annalisa; Mosci, Sofia; Rosasco, Lorenzo; Verri, Alessandro; Varesio, Luigi

    2009-10-15

    Gene expression signatures are clusters of genes discriminating different statuses of the cells and their definition is critical for understanding the molecular bases of diseases. The identification of a gene signature is complicated by the high dimensional nature of the data and by the genetic heterogeneity of the responding cells. The l1-l2 regularization is an embedded feature selection technique that fulfills all the desirable properties of a variable selection algorithm and has the potential to generate a specific signature even in biologically complex settings. We studied the application of this algorithm to detect the signature characterizing the transcriptional response of neuroblastoma tumor cell lines to hypoxia, a condition of low oxygen tension that occurs in the tumor microenvironment. We determined the gene expression profile of 9 neuroblastoma cell lines cultured under normoxic and hypoxic conditions. We studied a heterogeneous set of neuroblastoma cell lines to mimic the in vivo situation and to test the robustness and validity of the l1-l2 regularization with double optimization. Analysis by hierarchical, spectral, and k-means clustering or supervised approach based on t-test analysis divided the cell lines on the bases of genetic differences. However, the disturbance of this strong transcriptional response completely masked the detection of the more subtle response to hypoxia. Different results were obtained when we applied the l1-l2 regularization framework. The algorithm distinguished the normoxic and hypoxic statuses defining signatures comprising 3 to 38 probesets, with a leave-one-out error of 17%. A consensus hypoxia signature was established setting the frequency score at 50% and the correlation parameter epsilon equal to 100. This signature is composed by 11 probesets representing 8 well characterized genes known to be modulated by hypoxia. We demonstrate that l1-l2 regularization outperforms more conventional approaches allowing the

  16. Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Yanyan; Verbiest, Tom; Devery, Aoife M.; Bokobza, Sivan M.; Weber, Anika M.; Leszczynska, Katarzyna B.; Hammond, Ester M.; Ryan, Anderson J., E-mail: anderson.ryan@oncology.ox.ac.uk

    2016-06-01

    Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models. Methods and Materials: NSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O{sub 2}) or hypoxic (1% O{sub 2}) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, daily for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth. Results: In vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O{sub 2}). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors. Conclusions: Our data suggest that hypoxia potentiates the radiation-sensitizing effects of

  17. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kyotani, Yoji, E-mail: cd147@naramed-u.ac.jp [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Ota, Hiroyo [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Zhao, Jing; Ozawa, Kentaro; Nagayama, Kosuke; Ito, Satoyasu [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Takasawa, Shin [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Kimura, Hiroshi [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Uno, Masayuki [Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Yoshizumi, Masanori [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan)

    2013-11-15

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin m

  18. Hypoxia, Oxidative Stress and Fat

    Directory of Open Access Journals (Sweden)

    Nikolaus Netzer

    2015-06-01

    Full Text Available Metabolic disturbances in white adipose tissue in obese individuals contribute to the pathogenesis of insulin resistance and the development of type 2 diabetes mellitus. Impaired insulin action in adipocytes is associated with elevated lipolysis and increased free fatty acids leading to ectopic fat deposition in liver and skeletal muscle. Chronic adipose tissue hypoxia has been suggested to be part of pathomechanisms causing dysfunction of adipocytes. Hypoxia can provoke oxidative stress in human and animal adipocytes and reduce the production of beneficial adipokines, such as adiponectin. However, time-dose responses to hypoxia relativize the effects of hypoxic stress. Long-term exposure of fat cells to hypoxia can lead to the production of beneficial substances such as leptin. Knowledge of time-dose responses of hypoxia on white adipose tissue and the time course of generation of oxidative stress in adipocytes is still scarce. This paper reviews the potential links between adipose tissue hypoxia, oxidative stress, mitochondrial dysfunction, and low-grade inflammation caused by adipocyte hypertrophy, macrophage infiltration and production of inflammatory mediators.

  19. Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia.

    Science.gov (United States)

    Song, Seung; Park, Jong-Tae; Na, Joo Young; Park, Man-Seok; Lee, Jeong-Kil; Lee, Min-Cheol; Kim, Hyung-Seok

    2014-05-01

    Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3-7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular

  20. Hydrogen sulfide protects against chemical hypoxia-induced injury by inhibiting ROS-activated ERK1/2 and p38MAPK signaling pathways in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Aiping Lan

    Full Text Available Hydrogen sulfide (H(2S has been proposed as a novel neuromodulator and neuroprotective agent. Cobalt chloride (CoCl(2 is a well-known hypoxia mimetic agent. We have demonstrated that H(2S protects against CoCl(2-induced injuries in PC12 cells. However, whether the members of mitogen-activated protein kinases (MAPK, in particular, extracellular signal-regulated kinase1/2(ERK1/2 and p38MAPK are involved in the neuroprotection of H(2S against chemical hypoxia-induced injuries of PC12 cells is not understood. We observed that CoCl(2 induced expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α, decreased cystathionine-β synthase (CBS, a synthase of H(2S expression, and increased generation of reactive oxygen species (ROS, leading to injuries of the cells, evidenced by decrease in cell viability, dissipation of mitochondrial membrane potential (MMP , caspase-3 activation and apoptosis, which were attenuated by pretreatment with NaHS (a donor of H(2S or N-acetyl-L cystein (NAC, a ROS scavenger. CoCl(2 rapidly activated ERK1/2, p38MAPK and C-Jun N-terminal kinase (JNK. Inhibition of ERK1/2 or p38MAPK or JNK with kinase inhibitors (U0126 or SB203580 or SP600125, respectively or genetic silencing of ERK1/2 or p38MAPK by RNAi (Si-ERK1/2 or Si-p38MAPK significantly prevented CoCl(2-induced injuries. Pretreatment with NaHS or NAC inhibited not only CoCl(2-induced ROS production, but also phosphorylation of ERK1/2 and p38MAPK. Thus, we demonstrated that a concurrent activation of ERK1/2, p38MAPK and JNK participates in CoCl(2-induced injuries and that H(2S protects PC12 cells against chemical hypoxia-induced injuries by inhibition of ROS-activated ERK1/2 and p38MAPK pathways. Our results suggest that inhibitors of ERK1/2, p38MAPK and JNK or antioxidants may be useful for preventing and treating hypoxia-induced neuronal injury.

  1. The biochemical mechanism of hypoxia-induced mobilization of glycogen in cultured cancer cell

    OpenAIRE

    Mung, KL; Wong, NS

    2014-01-01

    BACKGROUND: Metabolic reprogramming is one of the strategies adopted by cancer cells to survive hypoxic conditions. Recent findings suggest that hypoxic cancer cells derive the energy that they need through glycolysis using glucose mobilized from intracellular glycogen reserve. Glycogen phosphorylase (GP) is the major rate-determining enzyme for glycogen mobilization in many normal cells under the condition of starvation or physical exercise. The lysosomal alpha-glucosidase (GAA) has also bee...

  2. CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes

    NARCIS (Netherlands)

    Hasegawa, Yuki; Tang, Dave; Takahashi, Naoko; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Suzuki, Harukazu; Kawaji, Hideya; Rehli, Michael; Baillie, J. Kenneth; de Hoon, Michiel J. L.; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V.; Lizio, Marina; Itoh, Masayoshi; Andersson, Robin; Mungall, Christopher J.; Meehan, Terrence F.; Schmeier, Sebastian; Bertin, Nicolas; Jørgensen, Mette; Dimont, Emmanuel; Arner, Erik; Schmidl, Christian; Schaefer, Ulf; Medvedeva, Yulia A.; Plessy, Charles; Vitezic, Morana; Severin, Jessica; Semple, Colin A.; Ishizu, Yuri; Young, Robert S.; Francescatto, Margherita; Alam, Intikhab; Albanese, Davide; Altschuler, Gabriel M.; Arakawa, Takahiro; Archer, John A. C.; Arner, Peter; Babina, Magda; Baker, Sarah; Balwierz, Piotr J.; Beckhouse, Anthony G.; Pradhan-Bhatt, Swati; Blake, Judith A.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Burroughs, A. Maxwell; Califano, Andrea; Cannistraci, Carlo V.; Carbajo, Daniel; Chen, Yun; Chierici, Marco; Ciani, Yari; Clevers, Hans C.; Dalla, Emiliano; Davis, Carrie A.; Detmar, Michael; Diehl, Alexander D.; Dohi, Taeko; Drabløs, Finn; Edge, Albert S. B.; Edinger, Matthias; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Fagiolini, Michela; Fairbairn, Lynsey; Fang, Hai; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Favorov, Alexander V.; Fisher, Malcolm E.; Frith, Martin C.; Fujita, Rie; Fukuda, Shiro; Furlanello, Cesare; Furuno, Masaaki; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gibson, Andrew; Gingeras, Thomas; Goldowitz, Daniel; Gough, Julian; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Harbers, Matthias; Harshbarger, Jayson; Hasegawa, Akira; Hashimoto, Takehiro; Herlyn, Meenhard; Hitchens, Kelly J.; Ho Sui, Shannan J.; Hofmann, Oliver M.; Hoof, Ilka; Hori, Fumi; Huminiecki, Lukasz; Iida, Kei; Ikawa, Tomokatsu; Jankovic, Boris R.; Jia, Hui; Joshi, Anagha; Jurman, Giuseppe; Kaczkowski, Bogumil; Kai, Chieko; Kaida, Kaoru; Kaiho, Ai; Kajiyama, Kazuhiro; Kanamori-Katayama, Mutsumi; Kasianov, Artem S.; Kasukawa, Takeya; Katayama, Shintaro; Kato, Sachi; Kawaguchi, Shuji; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kawashima, Tsugumi; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klinken, S. Peter; Knox, Alan J.; Kojima, Miki; Kojima, Soichi; Kondo, Naoto; Koseki, Haruhiko; Koyasu, Shigeo; Krampitz, Sarah; Kubosaki, Atsutaka; Kwon, Andrew T.; Laros, Jeroen F. J.; Lee, Weonju; Lennartsson, Andreas; Li, Kang; Lilje, Berit; Lipovich, Leonard; Mackay-sim, Alan; Manabe, Ri-ichiroh; Mar, Jessica C.; Marchand, Benoit; Mathelier, Anthony; Mejhert, Niklas; Meynert, Alison; Mizuno, Yosuke; Morais, David A. de Lima; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Motakis, Efthymios; Motohashi, Hozumi; Mummery, Christine L.; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nakazato, Kenichi; van Nimwegen, Erik; Ninomiya, Noriko; Nishiyori, Hiromi; Noma, Shohei; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohmiya, Hiroko; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Pain, Arnab; Passier, Robert; Patrikakis, Margaret; Persson, Helena; Piazza, Silvano; Prendergast, James G. D.; Rackham, Owen J. L.; Ramilowski, Jordan A.; Rashid, Mamoon; Ravasi, Timothy; Rizzu, Patrizia; Roncador, Marco; Roy, Sugata; Rye, Morten B.; Saijyo, Eri; Sajantila, Antti; Saka, Akiko; Sakaguchi, Shimon; Sakai, Mizuho; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schneider, Claudio; Schultes, Erik A.; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sengstag, Thierry; Sheng, Guojun; Shimoji, Hisashi; Shimoni, Yishai; Shin, Jay W.; Simon, Christophe; Sugiyama, Daisuke; Sugiyama, Takaaki; Suzuki, Masanori; Suzuki, Naoko; Swoboda, Rolf K.; 't Hoen, Peter A. C.; Tagami, Michihira; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tatum, Zuotian; Thompson, Mark; Toyoda, Hiroo; Toyoda, Tetsuro; Valen, Eivind; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Vorontsov, Ilya E.; Wasserman, Wyeth W.; Watanabe, Shoko; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Wood, Emily J.; Yamaguchi, Yoko; Yamamoto, Masayuki; Yoneda, Misako; Yonekura, Yohei; Yoshida, Shigehiro; Zabierowski, Suzan E.; Zhang, Peter G.; Zhao, Xiaobei; Zucchelli, Silvia; Summers, Kim M.; Daub, Carsten O.; Kawai, Jun; Heutink, Peter; Hide, Winston; Freeman, Tom C.; Lenhard, Boris; Bajic, Vladimir B.; Taylor, Martin S.; Makeev, Vsevolod J.; Sandelin, Albin; Hume, David A.; Carninci, Piero

    2014-01-01

    Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naive state ES which requires LIF to maintain pluripotency. Here we show that

  3. Expression of peptide NAP in rat retinal Müller cells prevents hypoxia-induced retinal injuries and promotes retinal neurons growth.

    Science.gov (United States)

    Zheng, Yuping; Zeng, Hao; She, Huaning; Liu, Hui; Sun, Naixue

    2010-07-01

    NAP (NAPVSIPQ) is a short peptide derived from activity-dependent neuroprotective protein (ADNP) sequence, whose potent and direct neuroprotective capabilities have been widely accepted. However, due to the high risk and inconvenience of intraocular injections, NAP is difficult to be clinically administered as therapeutic agent in treating retinal diseases. Currently, stable transfection of this octapeptide into cells has not been reported, partly because of its small size and lacking of 5' signal sequence. Here, we have developed a novel NT4-NAP fusion gene by attaching the 5' nonfunctional preproregion of neurotrophin 4 (NT4) to NAP cDNA. Recombinant adeno-associated virus was established to introduce NT4-NAP construct into cultured rat retinal Müller cells (RMC), resulting in sustained high level NAP production from stable transfection. Functional analyses of RMC cells transfected with NAP revealed the remarkably reduced cytotoxicity and apoptosis of the cells under hypoxia. Furthermore, coculturing of transfected RMC-NAP cells with primary rat retinal neural cells offer marked protection to the latter against hypoxia induced cellular damages. Together our data indicate that stable transfection of NAP into retinal Müller cells with constant NAP production is possible. NAP produced from cellular transfection maintained its biological neuroprotective activities. This targeted gene expression may provide an effective treatment for retinal diseases in the near future. Crown Copyright 2010. Published by Elsevier SAS. All rights reserved.

  4. Inhibition of Hypoxia Inducible Factor Alpha and Astrocyte-Elevated Gene-1 Mediates Cryptotanshinone Exerted Antitumor Activity in Hypoxic PC-3 Cells

    Directory of Open Access Journals (Sweden)

    Hyo-Jeong Lee

    2012-01-01

    Full Text Available Although cryptotanshinone (CT was known to exert antitumor activity in several cancers, its molecular mechanism under hypoxia still remains unclear. Here, the roles of AEG-1 and HIF-1α in CT-induced antitumor activity were investigated in hypoxic PC-3 cells. CT exerted cytotoxicity against prostate cancer cells and suppressed HIF-1α accumulation and AEG-1 expression in hypoxic PC-3 cells. Also, AEG-1 was overexpressed in prostate cancer cells. Interestingly, HIF-1α siRNA transfection enhanced the cleavages of caspase-9,3, and PAPR and decreased expression of Bcl-2 and AEG1 induced by CT in hypoxic PC-3 cells. Of note, DMOG enhanced the stability of AEG-1 and HIF-1α during hypoxia. Additionally, CT significantly reduced cellular level of VEGF in PC-3 cells and disturbed tube formation of HUVECs. Consistently, ChIP assay revealed that CT inhibited the binding of HIF-1α to VEGF promoter. Furthermore, CT at 10 mg/kg suppressed the growth of PC-3 cells in BALB/c athymic nude mice by 46.4% compared to untreated control. Consistently, immunohistochemistry revealed decreased expression of Ki-67, CD34, VEGF, carbonic anhydrase IX, and AEG-1 indices in CT-treated group compared to untreated control. Overall, our findings suggest that CT exerts antitumor activity via inhibition of HIF-1α, AEG1, and VEGF as a potent chemotherapeutic agent.

  5. Effects of YC-1 on hypoxia-inducible factor 1 alpha in hypoxic human bladder transitional carcinoma cell line T24 cells.

    Science.gov (United States)

    Li, Yangle; Zhao, Xiaokun; Tang, Huiting; Zhong, Zhaohui; Zhang, Lei; Xu, Ran; Li, Songchao; Wang, Yi

    2012-01-01

    It was the aim of this study to explore the effects of 3-(5'-hydroxymethyl-2'-furyl)-l-benzyl indazole (YC-1) on transcription activity, cell proliferation and apoptosis of hypoxic human bladder transitional carcinoma cells (BTCC), mediated by hypoxia-inducible factor 1α (HIF-1α). BTCC cell line T24 cells were incubated under normoxic or hypoxic conditions, adding different doses of YC-1. The protein expression of HIF-1α and HIF-1α-mediated genes was detected by Western blotting. RT-PCR was used to detect HIF-1α mRNA expression. Cell proliferation, apoptosis and migration activity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and transwell migration assay. The cells were pretreated by two ERK/p38 MAPK pathway-specific inhibitors, PD98059 or SB203580, and then incubated with YC-1 treatment under hypoxic condition. HIF-1α protein expression was detected by Western blotting. Hypoxic T24 cells expressed a higher level of HIF-1α, vascular endothelial growth factor, matrix metalloproteinases-2, B-cell lymphoma/leukemia-2 protein and HIF-1α mRNA compared with normoxic controls, in which the above-mentioned expression was downregulated by YC-1 in a dose-dependent manner. Cell proliferation and migration activity were inhibited while apoptosis was induced by YC-1 under hypoxic condition. Moreover, YC-1-downregulated HIF-1α expression was reversed by PD98059 and SB203580, respectively. YC-1 inhibits HIF-1α and HIF-1α-mediated gene expression, cell proliferation and migration activity and induces apoptosis in hypoxic BTCC. The ERK/p38 MAPK pathway may be involved in YC-1-mediated inhibition of HIF-1α. Copyright © 2011 S. Karger AG, Basel.

  6. Hypoxia-increased RAGE expression regulates chemotaxis and pro-inflammatory cytokines release through nuclear translocation of NF-κ B and HIF1α in THP-1 cells.

    Science.gov (United States)

    Bai, Wei; Zhou, Jing; Zhou, Na; Liu, Qin; Cui, Jian; Zou, Wei; Zhang, Wei

    2018-01-15

    The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1 cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H 2 O 2 or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1β, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1 cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H 2 O 2 . Moreover, SNP or H 2 O 2 exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1 cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1β. Thus, hypoxia not only increases RAGE expression in THP-1 cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Intermittent hypoxia and neurorehabilitation.

    Science.gov (United States)

    Gonzalez-Rothi, Elisa J; Lee, Kun-Ze; Dale, Erica A; Reier, Paul J; Mitchell, Gordon S; Fuller, David D

    2015-12-15

    In recent years, it has become clear that brief, repeated presentations of hypoxia [i.e., acute intermittent hypoxia (AIH)] can boost the efficacy of more traditional therapeutic strategies in certain cases of neurologic dysfunction. This hypothesis derives from a series of studies in animal models and human subjects performed over the past 35 yr. In 1980, Millhorn et al. (Millhorn DE, Eldridge FL, Waldrop TG. Respir Physiol 41: 87-103, 1980) showed that electrical stimulation of carotid chemoafferent neurons produced a persistent, serotonin-dependent increase in phrenic motor output that outlasts the stimulus for more than 90 min (i.e., a "respiratory memory"). AIH elicits similar phrenic "long-term facilitation" (LTF) by a mechanism that requires cervical spinal serotonin receptor activation and de novo protein synthesis. From 2003 to present, a series of studies demonstrated that AIH can induce neuroplasticity in the injured spinal cord, causing functional recovery of breathing capacity after cervical spinal injury. Subsequently, it was demonstrated that repeated AIH (rAIH) can induce recovery of limb function, and the functional benefits of rAIH are greatest when paired with task-specific training. Since uncontrolled and/or prolonged intermittent hypoxia can elicit pathophysiology, a challenge of intermittent hypoxia research is to ensure that therapeutic protocols are well below the threshold for pathogenesis. This is possible since many low dose rAIH protocols have induced functional benefits without evidence of pathology. We propose that carefully controlled rAIH is a safe and noninvasive modality that can be paired with other neurorehabilitative strategies including traditional activity-based physical therapy or cell-based therapies such as intraspinal transplantation of neural progenitors. Copyright © 2015 the American Physiological Society.

  8. Radiosensitization of normoxic and hypoxic h1339 lung tumor cells by heat shock protein 90 inhibition is independent of hypoxia inducible factor-1α.

    Science.gov (United States)

    Schilling, Daniela; Bayer, Christine; Li, Wei; Molls, Michael; Vaupel, Peter; Multhoff, Gabriele

    2012-01-01

    Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1α (HIF-1α). Overexpression of HIF-1α is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy. Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1α levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1α levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1α upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1α levels and a concomitant increase in radiosensitivity. In summary, our data show a HIF-1α-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition.

  9. Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1α and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Sun

    2017-12-01

    Full Text Available Background/Aims: The goal of this study was to detect the expression of hypoxia-inducible factor 1α (HIF-1α and vascular endothelial growth factor (VEGF in human retinal pigmented epithelial (RPE cells treated with celecoxib, a selective cyclooxygenase-2 (COX-2 inhibitor, under hypoxic and normoxic conditions and to explore the signaling mechanism involved in regulating the hypoxia-induced expression of HIF-1α and VEGF in RPE cells. Methods: D407 cells were cultured in normoxic or hypoxic conditions, with or without celecoxib or a PI3K inhibitor (LY294002. The anti-proliferative effect of celecoxib was assessed using the MTT assay. RT-PCR, Western blotting and ELISA were performed to detect the levels of PI3K, phosphorylated AKT (p-AKT, HIF-1α, VEGF and COX-2. Results: Celecoxib inhibited the proliferation of RPE cells in a dose-dependent manner. Celecoxib suppressed the expression of VEGF at both the mRNA and protein levels and decreased HIF-1α protein expression. HIF-1α activation was regulated by the PI3K/AKT pathway. The celecoxib-induced down-regulation of HIF-1α and VEGF required the suppression of the hypoxia-induced PI3K/AKT pathway. However, the down-regulation of COX-2 did not occur in cells treated with celecoxib. Conclusions: The antiangiogenic effects of celecoxib in RPE cells under hypoxic conditions resulted from the inhibition of HIF-1α and VEGF expression, which may be partly mediated by a COX-2-independent, PI3K/AKT-dependent pathway.

  10. Activation of PLA2 isoforms by cell swelling and ischaemia/hypoxia

    DEFF Research Database (Denmark)

    Lambert, I. H.; Pedersen, S. F.; Poulsen, K. A.

    2006-01-01

    Phospholipase A2 (PLA2) activity is increased in mammalian cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid....... Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the cell type and the physiological stress condition....

  11. Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

    Science.gov (United States)

    Cammarata, Patrick R; Neelam, Sudha; Brooks, Morgan M

    2015-01-01

    The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. Recently, we reported

  12. Hypoxia-increased RAGE and P2X7R expression regulates tumor cell invasion through phosphorylation of Erk1/2 and Akt and nuclear translocation of NF-{kappa}B.

    Science.gov (United States)

    Tafani, Marco; Schito, Luana; Pellegrini, Laura; Villanova, Lidia; Marfe, Gabriella; Anwar, Tahira; Rosa, Roberta; Indelicato, Manuela; Fini, Massimo; Pucci, Bruna; Russo, Matteo A

    2011-08-01

    The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.

  13. Proteomic analysis of hydroxyl radical-induced resuscitation of hypoxia-induced dormant mycobacterial cells

    Directory of Open Access Journals (Sweden)

    Abhishek Mishra

    2018-01-01

    Full Text Available Background: The genus Mycobacterium has an ability to persist in hostile environments for years before its reactivation in favorable conditions. The major bottleneck in decades of mycobacterial research is a poor understanding of molecular mechanism which assists bacteria to attain dormancy and reactivation later. Methods: In this study, hydroxyl radical was quantified in aerobically growing mycobacterial cells us