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Sample records for rapamycin inhibited hypoxia-induced

  1. The anti-diabetic drug metformin inhibits vascular endothelial growth factor expression via the mammalian target of rapamycin complex 1/hypoxia-inducible factor-1α signaling pathway in ELT-3 cells.

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    Tadakawa, Mari; Takeda, Takashi; Li, Bin; Tsuiji, Kenji; Yaegashi, Nobuo

    2015-01-05

    The aim of this study was to elucidate whether metformin can regulate the expression of vascular endothelial growth factor (VEGF) in rat-derived uterine leiomyoma cells (ELT-3 cells). In vitro studies were conducted using ELT-3 cells. Under normoxic conditions, metformin suppressed VEGF protein levels in the supernatant and cells in a dose-dependent manner. In hypoxia-mimicking conditions, VEGF and hypoxia-inducible factor-1α (HIF-1α) proteins were both highly expressed and were suppressed by the metformin treatment. Metformin did not affect HIF-1α mRNA levels, which indicated that its effects occurred at the post-translational level. Metformin inhibited mammalian target of rapamycin complex 1 (mTORC1) activity by phosphorylating the mTORC1 component raptor. This study revealed the anti-angiogenic activity of metformin in ELT-3 cells by suppressing the expression of VEGF via the mTORC1/HIF-1α pathway. These results indicate that metformin may represent an effective alternative in the future treatment of uterine leiomyomas. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

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    Thiepold, Anna-Luisa; Lorenz, Nadja I; Foltyn, Martha; Engel, Anna L; Divé, Iris; Urban, Hans; Heller, Sonja; Bruns, Ines; Hofmann, Ute; Dröse, Stefan; Harter, Patrick N; Mittelbronn, Michel; Steinbach, Joachim P; Ronellenfitsch, Michael W

    2017-10-01

    Glioblastomas are characterized by fast uncontrolled growth leading to hypoxic areas and necrosis. Signalling from EGFR via mammalian target of rapamycin complex 1 (mTORC1) is a major driver of cell growth and proliferation and one of the most commonly altered signalling pathways in glioblastomas. Therefore, epidermal growth factor receptor and mTORC1 signalling are plausible therapeutic targets and clinical trials with inhibitors are in progress. However, we have previously shown that epidermal growth factor receptor and mTORC1 inhibition triggers metabolic changes leading to adverse effects under the conditions of the tumour microenvironment by protecting from hypoxia-induced cell death. We hypothesized that conversely mTORC1 activation sensitizes glioma cells to hypoxia-induced cell death. As a model for mTORC1 activation we used gene suppression of its physiological inhibitor TSC2 (TSC2sh). TSC2sh glioma cells showed increased sensitivity to hypoxia-induced cell death that was accompanied by an earlier ATP depletion and an increase in reactive oxygen species. There was no difference in extracellular glucose consumption but an altered intracellular metabolic profile with an increase of intermediates of the pentose phosphate pathway. Mechanistically, mTORC1 upregulated the first and rate limiting enzyme of the pentose phosphate pathway, G6PD. Furthermore, an increase in oxygen consumption in TSC2sh cells was detected. This appeared to be due to higher transcription rates of genes involved in mitochondrial respiratory function including PPARGC1A and PPARGC1B (also known as PGC-1α and -β). The finding that mTORC1 activation causes an increase in oxygen consumption and renders malignant glioma cells susceptible to hypoxia and nutrient deprivation could help identify glioblastoma patient cohorts more likely to benefit from hypoxia-inducing therapies such as the VEGFA-targeting antibody bevacizumab in future clinical evaluations. © The Author (2017). Published by

  3. Rapamycin attenuates hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy in mice

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    Tillmanns Harald H

    2007-02-01

    Full Text Available Abstract Background Chronic hypoxia induces pulmonary arterial hypertension (PAH. Smooth muscle cell (SMC proliferation and hypertrophy are important contributors to the remodeling that occurs in chronic hypoxic pulmonary vasculature. We hypothesized that rapamycin (RAPA, a potent cell cycle inhibitor, prevents pulmonary hypertension in chronic hypoxic mice. Methods Mice were held either at normoxia (N; 21% O2 or at hypobaric hypoxia (H; 0.5 atm; ~10% O2. RAPA-treated animals (3 mg/kg*d, i.p. were compared to animals injected with vehicle alone. Proliferative activity within the pulmonary arteries was quantified by staining for Ki67 (positive nuclei/vessel and media area was quantified by computer-aided planimetry after immune-labeling for α-smooth muscle actin (pixel/vessel. The ratio of right ventricle to left ventricle plus septum (RV/[LV+S] was used to determine right ventricular hypertrophy. Results Proliferative activity increased by 34% at day 4 in mice held under H (median: 0.38 compared to N (median: 0.28, p = 0.028 which was completely blocked by RAPA (median HO+RAPA: 0.23, p = 0.003. H-induced proliferation had leveled off within 3 weeks. At this time point media area had, however, increased by 53% from 91 (N to 139 (H, p Conclusion Therapy with rapamycin may represent a new strategy for the treatment of pulmonary hypertension.

  4. Galectin-3 inhibition ameliorates hypoxia-induced pulmonary artery hypertension.

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    Hao, Mingwen; Li, Miaomiao; Li, Wenjun

    2017-01-01

    Galectin-3 (Gal-3) is a β-galactoside-binding lectin, which is important in inflammation, fibrosis and heart failure. The present study aimed to investigate the role and mechanism of Gal-3 in hypoxia-induced pulmonary arterial hypertension (PAH). Male C57BL/6J and Gal‑3‑/‑ mice were exposed to hypoxia, then the right ventricular systolic pressure (RVSP) and Fulton's index were measured, and Gal‑3 mRNA and protein expression in the pulmonary arteries was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blotting. Compared with the control, hypoxia increased the mRNA and protein expression levels of Gal‑3 in wild type murine pulmonary arteries. Gal‑3 deletion reduced the hypoxia‑induced upregulation of RVSP and Fulton's index. Furthermore, human pulmonary arterial endothelial cells (HPAECs) and human pulmonary arterial smooth muscle cells (HPASMCs) were stimulated by hypoxia in vitro, and Gal‑3 expression was inhibited by small interfering RNA. The inflammatory response of HPAECs, and the proliferation and cell cycle distribution of HPASMCs was also analyzed. Gal‑3 inhibition alleviated the hypoxia‑induced inflammatory response in HPAECs, including tumor necrosis factor‑α and interleukin‑1 secretion, expression of intercellular adhesion molecule‑1 and adhesion of THP‑1 monocytes. Gal‑3 inhibition also reduced hypoxia‑induced proliferation of HPASMCs, partially by reducing cyclin D1 expression and increasing p27 expression. Furthermore, Gal‑3 inhibition suppressed HPASMC switching from a 'contractile' to a 'synthetic' phenotype. In conclusion, Gal‑3 serves a fundamental role in hypoxia‑induced PAH, and inhibition of Gal‑3 may represent a novel therapeutic target for the treatment of hypoxia-induced PAH.

  5. DPP-4 inhibition protects human umbilical vein endothelial cells from hypoxia-induced vascular barrier impairment

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    Naoko Hashimoto

    2017-09-01

    Full Text Available Dipeptidyl peptidase-4 (DPP-4 inhibitors are relatively new class of anti-diabetic drugs. Some protective effects of DPP-4 on cardiovascular disease have been described independently from glucose-lowering effect. However, the detailed mechanisms by which DPP-4 inhibitors exert on endothelial cells remain elusive. The purpose of this research was to determine the effects of DPP-4 inhibitor on endothelial barrier function. Human umbilical vein endothelial cells (HUVECs were cultured and exposed to hypoxia in the presence or absence of Diprotin A, a DPP-4 inhibitor. Immunocytochemistry of vascular endothelial (VE- cadherin showed that jagged VE-cadherin staining pattern induced by hypoxia was restored by treatment with Diprotin A. The increased level of cleaved β-catenin in response to hypoxia was significantly attenuated by Diprotin A, suggesting that DPP-4 inhibition protects endothelial adherens junctions from hypoxia. Subsequently, we found that Diprotin A inhibited hypoxia-induced translocation of NF-κB from cytoplasm to nucleus through decreasing TNF-α expression level. Furthermore, the tube formation assay showed that Diprotin A significantly restored hypoxia-induced decrease in number of tubes by HUVECs. These results suggest that DPP-4 inhibitior protects HUVECs from hypoxia-induced barrier impairment.

  6. Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1α/VEGF-A Pathway in Colorectal Cancer

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    Hongwei Chen

    2015-01-01

    Full Text Available Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway.

  7. Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation

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    Park, Choa; Lee, YoungJoo, E-mail: yjlee@sejong.ac.kr

    2014-07-18

    Highlights: • We examined the effect of ERβ specific ligand on HIF-1 inhibition. • DPN down-regulates the ARNT protein levels in PC3 cells. • DPN did not show additional effect in ERβ transfected MCF-7 cells. • Our study shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression. - Abstract: Estrogen receptor (ER) β is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERβ suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERβ specific ligand on HIF-1 inhibition in ERβ positive PC3 cells and ERβ transfected MCF-7 cells. ERβ specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERβ specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERβ transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERβ was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERβ significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERβ. This result shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression.

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

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    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.

  9. Inactivation of lysyl oxidase by β-aminopropionitrile inhibits hypoxia-induced invasion and migration of cervical cancer cells.

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    Yang, Xiaoxiao; Li, Shifeng; Li, Wande; Chen, Jingkao; Xiao, Xiao; Wang, Youqiong; Yan, Guangmei; Chen, Lijun

    2013-02-01

    Tumor invasion and migration are major causes of mortality in patients with cervical carcinoma. Tumors under hypoxic conditions are more invasive and have a higher metastasic activity. Lysyl oxidase (LOX) is a hypoxia-responsive gene. LOX has been shown to be essential for hypoxia-induced metastasis in breast cancer. However, the direct impact of LOX on cervical cancer cell motility remains poorly understood. Our study revealed that LOX expression at protein and catalytic levels is upregulated in cervical cancer cells upon exposure to hypoxia. Hypoxia induced mesenchymal-like morphological changes in HeLa and SiHa cells which were accompanied by upregulation of α-SMA and vimentin, two mesenchymal markers, and downregulation of E-cadherin, an epithelial marker, indicating the epithelial-mesenchymal transition (EMT) of cervical cancer cells occurred under hypoxic conditions. Treatment of tumor cells with β-aminopropionitrile (BAPN), an active site inhibitor of LOX, blocked the hypoxia-induced EMT morphological and marker protein changes, and inhibited invasion and migration capacities of cervical carcinoma cells in vitro. Collectively, these findings suggest LOX enhances hypoxia-induced invasion and migration in cervical cancer cells mediated by the EMT which can be inhibited by BAPN.

  10. Brazilian Green Propolis Suppresses the Hypoxia-Induced Neuroinflammatory Responses by Inhibiting NF-κB Activation in Microglia

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    Zhou Wu

    2013-01-01

    Full Text Available Hypoxia has been recently proposed as a neuroinflammatogen, which drives microglia to produce proinflammatory cytokines, including interleukin-1β (IL-1β, tumor necrosis factor-α (TNF-α, and IL-6. Considering the fact that propolis has hepatoprotective, antitumor, antioxidative, and anti-inflammatory effects, propolis may have protective effects against the hypoxia-induced neuroinflammatory responses. In this study, propolis (50 μg/mL was found to significantly inhibit the hypoxia-induced cytotoxicity and the release of proinflammatory cytokines, including IL-1β, TNF-α, and IL-6, by MG6 microglia following hypoxic exposure (1% O2, 24 h. Furthermore, propolis significantly inhibited the hypoxia-induced generation of reactive oxygen species (ROS from mitochondria and the activation of nuclear factor-κB (NF-κB in microglia. Moreover, systemic treatment with propolis (8.33 mg/kg, 2 times/day, i.p. for 7 days significantly suppressed the microglial expression of IL-1β, TNF-α, IL-6, and 8-oxo-deoxyguanosine, a biomarker for oxidative damaged DNA, in the somatosensory cortex of mice subjected to hypoxia exposure (10% O2, 4 h. These observations indicate that propolis suppresses the hypoxia-induced neuroinflammatory responses through inhibition of the NF-κB activation in microglia. Furthermore, increased generation of ROS from the mitochondria is responsible for the NF-κB activation. Therefore, propolis may be beneficial in preventing hypoxia-induced neuroinflammation.

  11. Potent inhibition of tumoral hypoxia-inducible factor 1α by albendazole

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    Poruchynsky Marianne S

    2010-04-01

    Full Text Available Abstract Background Emerging reports suggest resistance, increased tumor invasiveness and metastasis arising from treatment with drugs targeting vascular endothelial growth factor (VEGF. It is believed that increased tumoral hypoxia plays a prominent role in the development of these phenomena. Inhibition of tumoral hypoxia inducible factor (HIF-1α is thus becoming an increasingly attractive therapeutic target in the treatment of cancer. We hypothesized that the anti-VEGF effect of albendazole (ABZ could be mediated through inhibition of tumoral HIF-1α. Method In vitro, the effects of ABZ on HIF-1α levels in human ovarian cancer cells (OVCAR-3 were investigated using hypoxic chamber or desferrioxamine (DFO induced-hypoxia. In vivo, the effects of ABZ (150 mg/kg, i.p., single dose on the tumor levels of HIF-1α and VEGF protein and mRNA were investigated by western blotting, RT-PCR and real time-PCR. Results In vitro, ABZ inhibited cellular HIF-1α protein accumulation resulting from placement of cells under hypoxic chamber or exposure to DFO. In vivo, tumors excised from vehicle treated mice showed high levels of both HIF-1α and VEGF. Whereas, tumoral HIF-1α and VEGF protein levels were highly suppressed in ABZ treated mice. Tumoral VEGFmRNA (but not HIF-1αmRNA was also found to be highly suppressed by ABZ. Conclusion These results demonstrate for the first time the effects of an acute dose of ABZ in profoundly suppressing both HIF-1α and VEGF within the tumor. This dual inhibition may provide additional value in inhibiting angiogenesis and be at least partially effective in inhibiting tumoral HIF-1α surge, tumor invasiveness and metastasis.

  12. Gingerol-induced hypoxia-inducible factor 1 alpha inhibits human prion peptide-mediated neurotoxicity.

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    Jeong, Jae-Kyo; Moon, Myung-Hee; Park, Yang-Gyu; Lee, Ju-Hee; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2013-08-01

    Prion diseases are a family member of neurodegenerative disorders caused by the accumulation of misfolded-prion proteins (scrapie form of PrP, PrP(Sc)). The accumulation of PrP(Sc) in the brain leads to neurotoxicity by the induction of mitochondrial-apoptotic pathways. Recent studies implicated gingerol in protection against neurodegeneration. However, the basis of the neuroprotection in prion disease remains unclear. Thus, we investigated the influence of gingerol on prion peptide-induced neuronal damage. Gingerol blocked PrP(106-126)-mediated neurotoxicity by protecting mitochondrial function. Moreover, the protective effect of gingerol against PrP(106-126)-induced mitochondrial damage was associated with hypoxia-inducible factor 1 alpha (HIF-1α) expression. Gingerol-induced HIF-1α expression inhibited the PrP(106-126)-induced mitochondrial dysfunction. On the other hand, inhibition of gingerol-induced HIF-1 α expression attenuated the gingerol-mediated neuroprotective effect. Here, we demonstrate for the first time that treatment with gingerol prevents prion peptide-mediated neuronal cell death and that the neuroprotection is induced by HIF-1α-mediated signals. This study suggests that treatment with gingerol may provide a novel therapeutic strategy for prion-mediated neurotoxicity. Copyright © 2012 John Wiley & Sons, Ltd.

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

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    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.

  14. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

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    Weijer, R.; Broekgaarden, M.; Krekorian, M.; Alles, L.K.; van Wijk, A.C; Mackaaij, C.; Verheij, J.; van der Wal, A.C.; van Gullik, T.M.; Storm, Gerrit; Heger, M.

    2016-01-01

    Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression

  15. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

    NARCIS (Netherlands)

    Weijer, Ruud; Broekgaarden, Mans; Krekorian, Massis; Alles, Lindy K.; van Wijk, Albert C.; Mackaaij, Claire; Verheij, Joanne; van der Wal, Allard C.; van Gulik, Thomas M.; Storm, Gert; Heger, Michal

    2016-01-01

    Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of

  16. Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

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    Burrows, Natalie; Cane, Gaelle; Robson, Mathew; Gaude, Edoardo; Howat, William J; Szlosarek, Peter W; Pedley, R Barbara; Frezza, Christian; Ashcroft, Margaret; Maxwell, Patrick H

    2016-03-14

    The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.

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

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    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

  18. The hypoxia-induced facilitation of augmented breaths is suppressed by the common effect of carbonic anhydrase inhibition.

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    Bell, Harold J; Haouzi, Philippe

    2010-05-31

    The typical respiratory response to hypoxia includes a dramatic facilitation of augmented breaths (ABs) or 'sighs' in the breathing rhythm. We recently found that when acetazolamide treatment is used to promote CO(2) retention and counteract alkalosis during exposure to hypoxia, then the hypoxia-induced facilitation of ABs is effectively prevented. These results indicate that hyperventilation-induced hypocapnia/alkalosis is an essential factor involved in the hypoxia-induced facilitation of augmented breaths. However, acetazolamide is also known to decrease the sensitivity of the arterial chemoreceptors. Therefore, the question remains as to whether acetazolamide prevents the facilitation of ABs during hypoxia by offsetting the effects of respiratory alkalosis, or alternatively by suppressing carotid body afferent activity. In the present study, we addressed this question by studying the effects of treatment with an alternative carbonic anhydrase inhibitor, methazolamide, which has been reported to leave carotid body responsiveness to hypoxia intact. Respiratory variables were monitored before, during and after 2 days of methazolamide treatment (10 mg kg(-1) IP, bid) in unsedated and unrestrained adult male rats. Pre-treatment, the number of ABs observed in a 5 min observation window was 1.2 + or - 0.8 and 17.4 + or - 3.8 in room air and hypoxia, respectively. During methazolamide treatment, the facilitation of ABs in hypoxia was rapidly and reversibly suppressed such that ABs we no longer significantly more frequent than they were in room air. The present results demonstrate that the hypoxia-induced facilitation of ABs can be suppressed via the general effects of carbonic anhydrase inhibition, which are common to both acetazolamide and methazolamide. We discuss these results as they pertain to the mechanisms regulating augmented breath production, and the possible association between hypocapnia/alkalosis and sleep disordered breathing. Copyright 2010 Elsevier B

  19. A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

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    Chai, Xiqing; Kong, Weina; Liu, Lingyun; Yu, Wenguo; Zhang, Zhenqing; Sun, Yimin

    2014-01-01

    Hypoxia-inducible factor 1 (HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we constructed a recombinant adeno-associated virus (rAAV) vector expressing the human HIF-1α gene (rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1α represses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results confirmed that rAAV-HIF-1α significantly reduces apoptosis induced by amyl...

  20. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

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    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  1. [Salidroside inhibits hypoxia-induced phenotypic modulation of corpus cavernosum smooth muscle cells in vitro].

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    Chen, Gang; Huang, Xiao-Jun; Lü, Bo-Dong; Chen, Shi-Tao; Zhang, Shi-Geng; Yang, Ke-Bing

    2013-08-01

    To explore the effects of salidroside on the phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMC) in hypoxic SD rats. CCSMCs were cultured in vitro and identified by immunohistochemistry. The cells were divided into six groups: normal control (21% O2), hypoxia (1% O2), hypoxia + salidroside 1 mg/L, hypoxia + salidroside 3 mg/L, hypoxia + salidroside 5 mg/L and hypoxia + PGE1 0.4 microg/L, and then cultured for 48 hours. The relative expressions of alpha-actin and osteopontin (OPN) in each group were determined by RT-PCR. The in vitro cultured CCSMCs grew well, with anti-alpha-smooth muscle actin monoclonal antibodies immunohistochemically positive. The relative expression of alpha-actin was markedly decreased while that of OPN remarkably increased in the hypoxia group as compared with the normal control group (P salidroside 5 mg/L group showed a significantly higher expression of alpha-actin and lower expression of OPN than the hypoxia group (P 0.05). Hypoxia can reduce the relative expression level of alpha-actin and increase that of OPN in the CCSMCs of SD rats, namely, induce their phenotypic modulation from the contraction to the non-contraction type. Salidroside can restrain hypoxia-induced phenotypic modulation of CCSMCs, and its inhibitory effect at 5 mg/L is similar to that of PGE1.

  2. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

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    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  3. Inhibition of Mammalian Target of Rapamycin Signaling with Rapamycin Prevents Trauma-Induced Heterotopic Ossification.

    Science.gov (United States)

    Qureshi, Ammar T; Dey, Devaveena; Sanders, Erin M; Seavey, Jonathan G; Tomasino, Allison M; Moss, Kaitlyn; Wheatley, Benjamin; Cholok, David; Loder, Shawn; Li, John; Levi, Benjamin; Davis, Thomas A

    2017-11-01

    A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1α by inhibiting prolyl-4-hydroxylase PHD2.

    Directory of Open Access Journals (Sweden)

    Peifeng Hou

    Full Text Available Hypoxia-inducible factor 1α (HIF-1α, a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1α is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA or fumarate hydratase (FH expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is not well documented. Here, we show that dimethyl-2-ketoglutarate (DKG, a cell membrane-permeable precursor of a key metabolic intermediate, α-ketoglutarate (α-KG, known for its ability to rescue glutamine deficiency, transiently stabilized HIF-1α by inhibiting activity of the HIF prolyl hydroxylase domain-containing protein, PHD2. Consequently, prolonged DKG-treatment under normoxia elevated HIF-1α abundance and up-regulated the expression of its downstream target genes, thereby inducing a pseudohypoxic condition. This HIF-1α stabilization phenotype is similar to that from treatment of cells with desferrioxamine (DFO, an iron chelator, or dimethyloxalyglycine (DMOG, an established PHD inhibitor, but was not recapitulated with other α-KG analogues, such as Octyl-2KG, MPTOM001 and MPTOM002. Our study is the first example of an α-KG precursor to increase HIF-1α abundance and activity. We propose that DKG acts as a potent HIF-1α activator, highlighting the potential use of DKG to investigate the contribution of PHD2-HIF-1α pathway to tumor biology.

  5. Rapamycin

    OpenAIRE

    Srivastava, Rupesh K.; Utley, Adam; Shrikant, Protul A.

    2012-01-01

    Vaccines that generate Ag-specific CD8+ T-cell responses of appropriate quality, magnitude and duration are highly desirable. The ability of mTOR to regulate CD8+ T-cell functional differentiation must be exploited for clinical benefit. In a recent paper, we report that varying the regimen of rapamycin administration regulates viral vaccine-induced CD8+ T-cell responses for tumor immunity. These observations validate the use of rapamycin in vaccination strategies and demonstrate the efficacy ...

  6. Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative mechanism.

    Science.gov (United States)

    Cavasin, Maria A; Demos-Davies, Kim; Horn, Todd R; Walker, Lori A; Lemon, Douglas D; Birdsey, Nicholas; Weiser-Evans, Mary C M; Harral, Julie; Irwin, David C; Anwar, Adil; Yeager, Michael E; Li, Min; Watson, Peter A; Nemenoff, Raphael A; Buttrick, Peter M; Stenmark, Kurt R; McKinsey, Timothy A

    2012-03-02

    Histone deacetylase (HDAC) inhibitors are efficacious in models of hypertension-induced left ventricular heart failure. The consequences of HDAC inhibition in the context of pulmonary hypertension with associated right ventricular cardiac remodeling are poorly understood. This study was performed to assess the utility of selective small-molecule inhibitors of class I HDACs in a preclinical model of pulmonary hypertension. Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs 1, 2, and 3. The compound reduced pulmonary arterial pressure more dramatically than tadalafil, a standard-of-care therapy for human pulmonary hypertension that functions as a vasodilator. MGCD0103 improved pulmonary artery acceleration time and reduced systolic notching of the pulmonary artery flow envelope, which suggests a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced pulmonary arterial pressure in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening because of suppression of smooth muscle cell proliferation. Right ventricular function was maintained in MGCD0103-treated animals. Although the class I HDAC inhibitor only modestly reduced right ventricular hypertrophy, it had multiple beneficial effects on the right ventricle, which included suppression of pathological gene expression, inhibition of proapoptotic caspase activity, and repression of proinflammatory protein expression. By targeting distinct pathogenic mechanisms, isoform-selective HDAC inhibitors have potential as novel therapeutics for pulmonary hypertension that will complement vasodilator standards of care.

  7. 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

    pathways, is associated with poor prognosis in many types of cancer. Therefore, down-regulation of HIF-1alpha protein by RNA antagonists may control cancer growth. EZN-2968 is a RNA antagonist composed of third-generation oligonucleotide, locked nucleic acid, technology that specifically binds and inhibits......-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...... with DU145 cells treated with EZN-2968. Ongoing phase I studies of EZN-2968 in patients with advanced malignancies will determine optimal dose and schedule for the phase II program....

  8. Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eun Joo [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Sowers, Anastasia; Thetford, Angela [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); McKay-Corkum, Grace; Chung, Su I. [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Mitchell, James B. [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Citrin, Deborah E., E-mail: citrind@mail.nih.gov [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States)

    2016-11-15

    Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials: C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results: Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P=.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P<.001). Conclusions: Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

  9. Minocycline ameliorates hypoxia-induced blood-brain barrier damage by inhibition of HIF-1α through SIRT-3/PHD-2 degradation pathway.

    Science.gov (United States)

    Yang, F; Zhou, L; Wang, D; Wang, Z; Huang, Q-Y

    2015-09-24

    Minocycline, a second-generation tetracycline alleviates neuro-inflammation and protects the blood-brain barrier (BBB) in ischemia stroke. However, the effect of minocycline in hypoxia-induced BBB damage is unclear. Here, we have investigated the effect of minocycline under hypoxia and explored its possible underlying mechanisms. The effect of minocycline was examined in vitro in Human Brain Microvascular Endothelial Cells (HBMECs) using Trans Epithelial Electric Resistance (TEER). Protein and mRNA expression of Hypoxia-Inducible Factors-1α (HIF-1α), matrix metalloproteinases (MMP-2 and MMP-9) and tight junction proteins (TJs) were detected by using Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The translocation and transcription of HIF-1α were detected by using immunocytochemistry and luciferase reporter assay. In vivo, to adult male Sprague Dawley (SD) rats under hypobaric hypoxia were administered minocycline for 1h and BBB permeability was tested by using Evans Blue and Transmission Electron Microscopy (TEM). Also, reduction of NAD-dependent deacetylase sirtuin-3 (SIRT-3)/proline hydroxylase-2 (PHD-2) signaling pathway was evaluated. Minocycline increased TEER in HBMECs after hypoxia (PMinocycline administration significantly reduced HIF-1α expression, protein and mRNA expression of MMP-2, MMP-9 and Vascular Endothelial Growth Factor (VEGF) (Pminocycline reversed the hypoxia-induced reduction of PHD-2 (Pminocycline were abolished by siRNA-mediated knockdown of SIRT-3 in the brain. Minocycline inhibits HIF-1α-mediated cellular responses and protects BBB integrity through SIRT-3/PHD-2 pathway, proving to be a potential drug for the prevention and treatment of hypoxic brain injuries. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Inhibition of vascular endothelial growth factor A and hypoxia-inducible factor 1α maximizes the effects of radiation in sarcoma mouse models through destruction of tumor vasculature.

    Science.gov (United States)

    Lee, Hae-June; Yoon, Changhwan; Park, Do Joong; Kim, Yeo-Jung; Schmidt, Benjamin; Lee, Yoon-Jin; Tap, William D; Eisinger-Mathason, T S Karin; Choy, Edwin; Kirsch, David G; Simon, M Celeste; Yoon, Sam S

    2015-03-01

    To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm(3) within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm(3) for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hae-June [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Yoon, Changhwan [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Park, Do Joong [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Department of Surgery, Seoul National University Bundang Hospital, Sungnam (Korea, Republic of); Kim, Yeo-Jung [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Schmidt, Benjamin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Lee, Yoon-Jin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Tap, William D. [Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Eisinger-Mathason, T.S. Karin [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Choy, Edwin [Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Kirsch, David G. [Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (United States); Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina (United States); Simon, M. Celeste [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Howard Hughes Medical Institute (United States); and others

    2015-03-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm{sup 3} within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm{sup 3} for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

  12. Inhibition of hemangioma growth using polymer-lipid hybrid nanoparticles for delivery of rapamycin.

    Science.gov (United States)

    Li, Haitao; Teng, Yunfei; Sun, Jin; Liu, Jianyong

    2017-11-01

    Although infantile hemangiomas is benign, its rapid growth may induce serious complications. However, only one drug Hemangeol™ has been approved by US Food and Drug Administration (FDA) to treat infantile hemangiomas. Thus it is necessary to develop novel alternative drugs to treat infantile hemangiomas. Rapamycin is a well-know potent antiangiogenic agent, whereas the daily oral administration of rapamycin exerts undesired metabolic effects due to its inhibition of mechanistic target of rapamycin (mTOR) which is critical in cell metabolism. We hereby developed rapamycin-loaded polymer-lipid hybrid nanoparticles (Rapamycin-PLNPs) as a local controlled release system to realize local and sustained release of rapamycin, aiming to reduce the side effects and frequency of administration of rapamycin. Rapamycin-PLNPs are of a small size (129.1nm), desired drug encapsulation efficiency (63.7%), and sustained drug release for 5 days. Rapamycin-PLNPs were shown to be able to effectively bind to hemangioma endothelia cells (HemECs), induce significant proliferation inhibition and reduce expression of angiogenesis factors in HemECs. The therapeutic effect of Rapamycin-PLNPs against infantile hemangioma in vivo was superior to rapamycin, as reflected by reduced hemangioma volume, weight and microvessel density. Taken together, Rapamycin-PLNPs represent a very promising local approach in the treatment of infantile hemangiomas. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Niclosamide enhances the antitumor effects of radiation by inhibiting the hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway in human lung cancer cells.

    Science.gov (United States)

    Xiang, Mei; Chen, Zihong; Yang, Donghong; Li, Haiwen; Zuo, Yufang; Li, Jingjing; Zhang, Wendian; Zhou, Hechao; Jiang, Danxian; Xu, Zumin; Yu, Zhonghua

    2017-08-01

    Lung cancer is one of the leading causes of cancer-associated mortality, worldwide. The overall survival rate remains low, but progress has been made in improving the diagnosis and treatment of lung cancer over the past decades. Niclosamide, a salicylanilide derivative used for the treatment of tapeworm infections, is safe, well tolerated, inexpensive and readily available. Previous studies have identified niclosamide as a potential anticancer agent. The present study demonstrated that niclosamide enhanced the effect of irradiation by inhibiting the hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway. These findings suggest that niclosamide may be a promising candidate for clinical evaluation as part of a combined regimen for the treatment of non-small cell lung cancer.

  14. Radiosensitization of normoxic and hypoxic h1339 lung tumor cells by heat shock protein 90 inhibition is independent of hypoxia inducible factor-1α.

    Directory of Open Access Journals (Sweden)

    Daniela Schilling

    Full Text Available 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.

  15. Sirtuin 6 Modulates Hypoxia-induced Apoptosis in Osteoblasts via Inhibition of Glycolysis: Implication for Pathogenesis of Periapical Lesions.

    Science.gov (United States)

    Kok, Sang-Heng; Hou, Kuo-Liang; Hong, Chi-Yuan; Chao, Ling-Hsiu; Hsiang-Hua Lai, Eddie; Wang, Han-Wei; Yang, Hsiang; Shun, Chia-Tung; Wang, Juo-Song; Lin, Sze-Kwan

    2015-10-01

    Osteoblast apoptosis is important in the regulation of inflammatory bone resorption. Hypoxia resulting from inflammation enhances glycolysis and apoptosis. Sirtuin 6 (SIRT6) is a modulator of glucose metabolism and apoptosis. In the study we assessed the role of SIRT6 in hypoxia-induced glycolysis and apoptosis in osteoblasts, with special attention on the significance of these cellular processes in periapical lesions. Human bone marrow-derived osteoblasts were cultured under hypoxia. Expression of lactate dehydrogenase A was examined by Western blot, and production of lactate was measured by colorimetric assay. Cleavage of poly (adenosine diphosphate ribose) polymerase was used as an apoptosis marker and assessed by Western blot. SIRT6 was overexpressed in osteoblasts by lentiviral gene transduction, and then glycolytic and apoptotic responses were studied. In a rat model of bacteria-induced periapical lesions, expressions of SIRT6 and markers of glycolysis and apoptosis in osteoblasts were examined. Hypoxia enhanced lactate dehydrogenase A expression and lactate production in osteoblasts. Poly (adenosine diphosphate ribose) polymerase cleavage was induced by hypoxia or lactate treatment. SIRT6 suppressed hypoxia-augmented glycolysis and inhibited apoptosis induced by hypoxia or lactate treatment. Expression of SIRT6 in osteoblasts was downregulated by hypoxia and inflammatory mediators. Development of periapical lesions in rats was associated with decreased expression of SIRT6 and increased glycolysis and apoptosis in osteoblasts. Our study suggested that hypoxia-induced apoptosis of osteoblasts is dependent on glycolytic activity. SIRT6 is a negative regulator of inflammation and may alleviate periapical lesions by suppressing osteoblastic glycolysis and apoptosis. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. Inhibition of mammalian target of rapamycin by rapamycin increases the radiosensitivity of esophageal carcinoma Eca109 cells

    OpenAIRE

    ZHANG, DEJUN; XIANG, JIE; GU, YUMING; XU, WEI; XU, HAO; ZU, MAOHENG; PEI, DONGSHENG; ZHENG, JUNNIAN

    2014-01-01

    The aim of the present study was to investigate whether radiation induces the mammalian target of rapamycin (Rap) (mTOR) signaling pathway in esophageal carcinoma Eca109 cells, and whether mTOR inhibition by rapamycin increases Eca109 cell radiosensitivity. Changes in the levels of mTOR signaling pathway and DNA damage-repair proteins in Eca109 cells prior to and following radiation were determined. The Eca109 cells were treated with Rap (0, 100, 200 and 400 nmol/l) in combination with radiat...

  18. Hypoxia-Inducible Hydrogels

    Science.gov (United States)

    Park, Kyung Min; Gerecht, Sharon

    2014-01-01

    Oxygen is vital for the existence of all multicellular organisms, acting as a signaling molecule regulating cellular activities. Specifically, hypoxia, which occurs when the partial pressure of oxygen falls below 5%, plays a pivotal role during development, regeneration, and cancer. Here we report a novel hypoxia-inducible (HI) hydrogel composed of gelatin and ferulic acid that can form hydrogel networks via oxygen consumption in a laccase-mediated reaction. Oxygen levels and gradients within the hydrogels can be accurately controlled and precisely predicted. We demonstrate that HI hydrogels guide vascular morphogenesis in vitro via hypoxia-inducible factors activation of matrix metalloproteinases and promote rapid neovascularization from the host tissue during subcutaneous wound healing. The HI hydrogel is a new class of biomaterials that may prove useful in many applications, ranging from fundamental studies of developmental, regenerative and disease processes through the engineering of healthy and diseased tissue models towards the treatment of hypoxia-regulated disorders. PMID:24909742

  19. A novel role for 3, 4-dichloropropionanilide (DCPA in the inhibition of prostate cancer cell migration, proliferation, and hypoxia-inducible factor 1alpha expression

    Directory of Open Access Journals (Sweden)

    Schafer Rosana

    2006-08-01

    Full Text Available Abstract Background The amide class compound, 3, 4-dichloropropionanilide (DCPA is known to affect multiple signaling pathways in lymphocyte and macrophage including the inhibition of NF-κB ability. However, little is known about the effect of DCPA in cancer cells. Hypoxia-inducible factor 1 (HIF-1 regulates the expression of many genes including vascular endothelial growth factor (VEGF, heme oxygenase 1, inducible nitric oxide synthase, aldolase, enolase, and lactate dehydrogenase A. HIF-1 expression is associated with tumorigenesis and angiogenesis. Methods We used Transwell assay to study cell migration, and used immunoblotting to study specific protein expression in the cells. Results In this report, we demonstrate that DCPA inhibited the migration and proliferation of DU145 and PC-3 prostate cancer cells induced by serum, insulin, and insulin-like growth factor I (IGF-I. We found that DCPA inhibited HIF-1 expression in a subunit-specific manner in these cancer cell lines induced by serum and growth factors, and decreased HIF-1α expression by affecting its protein stability. Conclusion DCPA can inhibit prostate cancer cell migration, proliferation, and HIF-1α expression, suggesting that DCPA could be potentially used for therapeutic purpose for prostate cancer in the future.

  20. Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

    Science.gov (United States)

    Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

    2016-01-01

    Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

  1. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

  2. MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

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    Zhang, Y; Guo, Y; Yang, C; Zhang, S; Zhu, X; Cao, L; Nie, W; Yu, H

    2017-01-01

    Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

  3. Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family.

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    Hewitson, Kirsty S; McNeill, Luke A; Riordan, Madeline V; Tian, Ya-Min; Bullock, Alex N; Welford, Richard W; Elkins, Jonathan M; Oldham, Neil J; Bhattacharya, Shoumo; Gleadle, Jonathan M; Ratcliffe, Peter J; Pugh, Christopher W; Schofield, Christopher J

    2002-07-19

    Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-alpha. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-alpha abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-alpha. Since FIH contains certain motifs present in iron- and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-alpha fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.

  4. Epidermal growth factor receptor inhibition reduces angiogenesis via hypoxia-inducible factor-1α and Notch1 in head neck squamous cell carcinoma.

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    Wei-Ming Wang

    Full Text Available Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC. The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.

  5. Rapamycin and mTORC1 Inhibition in the Mouse: Skin Cancer Prevention

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    Athar, Mohammad; Kopelovich, Levy

    2011-01-01

    Therapeutic and preventive effects of rapamycin include reduced risk of non-melanoma skin cancer (NMSC). In this issue of the journal (beginning on page XXX), Checkley et al. report that rapamycin inhibits mammalian target of rapamycin (mTOR) complex 1 in murine epidermis, thereby inhibiting tumor promotion mediated by tetradecanoyl phorbol-13 acetate (TPA) in association with a strong anti-inflammatory effect. Rapamycin is an immunosuppressive drug for preventing graft rejection in organ transplant recipients and reduces the risk of NMSC and Kaposi’s sarcoma in this population, albeit by mechanisms distinct from immunosuppression. Important future directions include identifying molecular predictors of rapamycin/rapalog sensitivity or resistance (potentially, for example, PI3K pathway alterations and KRAS mutations) and combined non-rapalog, mTOR-targeting approaches, all of which should increase efficacy and minimize toxicity. PMID:21733819

  6. Rapamycin inhibits spermatogenesis by changing the autophagy status through suppressing mechanistic target of rapamycin-p70S6 kinase in male rats.

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    Liu, Shangjing; Huang, Longxian; Geng, Yanqing; He, Junlin; Chen, Xuemei; Xu, Hao; Li, Rong; Wang, Yingxiong; Ding, Yubin; Liu, Xueqing

    2017-10-01

    Rapamycin (sirolimus) is an antiproliferative drug that has been widely used in the clinic as an immunosuppressant and a potential anticancer agent. Certain reports have indicated that rapamycin may induce male infertility through impairing sperm quality. The present study investigated the mechanism of male infertility caused by rapamycin and examined whether withdrawal of rapamycin could recover the number of sperm in rats. Male Sprague‑Dawley rats (n=100) were divided randomly into 5 groups: 3 rapamycin‑treated groups (2, 4 and 6 mg/kg) and 2 control groups [Blank and dimethyl sulfoxide (DMSO)]. Organ coefficients of the testes, number of sperm and hematoxylin‑eosin staining analyses demonstrated that rapamycin treatment markedly damaged the structure of the seminiferous tubule and reduced the number of sperm. Immunohistochemistry of mechanistic target of rapamycin (mTOR) and Ki67 in testes tissue, and western blotting of phosphorylated‑p70S6K and p70S6K, supported the hypothesis that rapamycin causes sperm reduction through inhibiting proliferation of spermatogonia. Unfortunately, 24 weeks after cessation of rapamycin treatment, only the number of sperm in 2 mg/kg group was restored back to the normal level. In addition, to the best of our knowledge, the present study was the first to demonstrate that low doses rapamycin leads to activation of autophagy in rat testes. This may be a self‑protective mechanism of the cell in response to external stress. Thus, spermatogenesis can be recovered in the testes from rats in the low dose group. High doses of rapamycin resulted in excessive consumption of autophagy proteins, and the damage could not be compensated. In addition, it was revealed that cell apoptosis increased after treatment with rapamycin. In conclusion, the present study demonstrated that rapamycin inhibits spermatogenesis through suppressing phosphorylation of p70S6K and changing the autophagy status, ultimately reducing the number of sperm

  7. A possible therapeutic potential of quercetin through inhibition of μ-calpain in hypoxia induced neuronal injury: a molecular dynamics simulation study

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    Anand Kumar Pandey

    2016-01-01

    Full Text Available The neuroprotective property of quercetin is well reported against hypoxia and ischemia in past studies. This property of quercetin lies in its antioxidant property with blood-brain barrier permeability and anti-inflammatory capabilities. µ-Calpain, a calcium ion activated intracellular cysteine protease causes serious cellular insult, leading to cell death in various pathological conditions including hypoxia and ischemic stroke. Hence, it may be considered as a potential drug target for the treatment of hypoxia induced neuronal injury. As the inhibitory property of µ-calpain is yet to be explored in details, hence, in the present study, we investigated the interaction of quercetin with µ-calpain through a molecular dynamics simulation study as a tool through clarifying the molecular mechanism of such inhibition and determining the probable sites and modes of quercetin interaction with the µ-calpain catalytic domain. In addition, we also investigated the structure-activity relationship of quercetin with μ-calpain. Affinity binding of quercetin with µ-calpain had a value of –28.73 kJ/mol and a Ki value of 35.87 µM that may be a probable reason to lead to altered functioning of µ-calpain. Hence, quercetin was found to be an inhibitor of µ-calpain which might have a possible therapeutic role in hypoxic injury.

  8. Rapamycin promotes β-amyloid production via ADAM-10 inhibition

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    Zhang, Sheqing; Salemi, Jon; Hou, Huayan; Zhu, Yuyan; Mori, Takashi; Giunta, Brian; Obregon, Demian; Tan, Jun

    2010-01-01

    Rapamycin is a well known immunosuppressant drug for rejection prevention in organ transplantation. Numerous clinical trials using rapamycin analogs, involving both children and adults with various disorders are currently ongoing worldwide. Most recently, rapamycin gained much attention for what appears to be life-span extending properties when administered to mice. The risk for Alzheimer disease (AD) is strongly and positively correlated with advancing age and is characterized by deposition of β-amyloid peptides (Aβ) as senile plaques in the brain. We report that rapamycin (2.5 μM), significantly increases Aβ generation in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP). In concert with these observations, we found rapamycin significantly decreases the neuroprotective amino-terminal APP (amyloid precursor protein) cleavage product, soluble APP-α (sAPP-α) while increasing production of the β-carboxyl-terminal fragment of APP (β-CTF). These cleavage events are associated with decreased activation of a disintegrin and metallopeptidase domain-10 (ADAM-10), an important candidate α-secretase which opposes Aβ generation. To validate these findings in vivo, we intraperitoneal (i.p.) injected Tg2576 Aβ-overproducing transgenic mice with rapamycin (3 mg/kg/day) for 2 weeks. We found increased Aβ levels associated with decreased sAPP-α at an average rapamycin plasma concentration of 169.7 ± 23.5 ng/mL by high performance liquid chromatography (HPLC). These data suggest that although rapamycin may increase the lifespan in some mouse models, it may not decrease the risk for age-associated neurodegenerative disorders such as AD. PMID:20542014

  9. Inhibition of MDM2 Re-Sensitizes Rapamycin Resistant Renal Cancer Cells via the Activation of p53.

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    Tian, Xin; Dai, Shundong; Sun, Jing; Jiang, Shenyi; Sui, Chengguang; Meng, Fandong; Li, Yan; Fu, Liye; Jiang, Tao; Wang, Yang; Su, Jia; Jiang, Youhong

    2016-01-01

    Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs. © 2016 S. Karger AG, Basel.

  10. [Silencing hypoxia inducible factor-2α gene by small interference RNA inhibits the growth of mammosphere cells in nude mice under hypoxic microenvironment].

    Science.gov (United States)

    Qu, Hong-bo; Fan, Yuan-ming; Han, Ming-li; Zeng, Ni; Zhu, Zhi-kun; Liu, Hong; Xie, Jia; Wu, Cheng-yi; Tang, Wei-xue

    2013-04-16

    To explore the effects of silencing hypoxia inducible factor-2α (HIF-2α) by small interference RNA on the growth of mammosphere cells in nude mice under hypoxic microenvironment. The empty and interference vectors were transfected into MCF-7 cell. Then G418 was added to screen the positive cells to obtain stable cell line. The empty and interference vectors were inoculated subcutaneously into left and right back near hind limb of nude mice. The volume and weight of tumors were calculated respectively. The expressions of HIF-2α, CD44, OCT-4 and KLF-4 protein in xenograft tumor tissues were detected by Western blot. The expression vector of HIF-2α-siRNA was successfully established. The formation of mammosphere was lowered by silencing HIF-2α gene expression. In contract to empty vector group cell, there were obvious decreases in the volumes and weights of tumors in interference group (P interference group (0.42 ± 0.01) was much lower than that of the empty vector group (0.89 ± 0.03, P interference group (0.21 ± 0.01) was much lower than the empty vector group (0.78 ± 0.03, P interference group (0.42 ± 0.01)was much lower than the empty vector group (0.68 ± 0.03, P interference group (0.34 ± 0.01) was much lower than the empty vector group (0.72 ± 0.03, P < 0.05). Silencing HIF-2α gene can effectively inhibit the growth of breast cancer stem cells in nude mice under hypoxic microenvironment. Its mechanism may be through inhibited capacity for self-renewal and proliferation of breast cancer stem cells in vivo through the down-regulated expressions of markers associated with stem cells. HIF-2α is expected to become a new target for gene therapy of breast cancer.

  11. Rapamycin-induced inhibition of HTLV-I LTR activity is rescued by c-Myb

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    Lever Andrew ML

    2007-04-01

    Full Text Available Abstract Background Rapamycin is an immunosuppressive which represses translation of transcripts harbouring a polypyrimidine motif downstream of the mRNA cap site through the mammalian target of rapamycin complex. It inhibits the abnormal autologous proliferation of T-cell clones containing a transcriptionally active human T-lymphotropic virus, type I (HTLV-I provirus, generated from infected subjects. We showed previously that this effect is independent of the polypyrimidine motifs in the viral long terminal repeat (LTR R region suggesting that HTLV-I transcription, and not translation, is being affected. Here we studied whether rapamycin is having an effect on a specific transcription factor pathway. Further, we investigated whether mRNAs encoding transcription factors involved in HTLV-I transcriptional activation, specifically CREB, Ets and c-Myb, are implicated in the rapamycin-sensitivity of the HTLV-I LTR. Results An in vitro analysis of the role of SRE- and NF-κB-mediated transcription highlighted the latter as rapamycin sensitive. Over-expression of c-Myb reversed the rapamycin effect. Conclusion The sensitivity of HTLV-I transcription to rapamycin may be effected through an NF-κB-pathway associated with the rapamycin-sensitive mTORC1 cellular signalling network.

  12. Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

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    Medici, Damian; Olsen, Bjorn R.

    2012-01-01

    Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas. PMID:22900063

  13. Rapamycin inhibits proliferation of hemangioma endothelial cells by reducing HIF-1-dependent expression of VEGF.

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    Damian Medici

    Full Text Available Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF signaling through VEGF receptor 2 (VEGFR2. Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1. VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.

  14. Inhibition of mammalian target of rapamycin reduces epileptogenesis and blood-brain barrier leakage but not microglia activation.

    NARCIS (Netherlands)

    van Vliet, E.A.; Forte, G.; Holtman, L.; den Burger, J.C.G.; Sinjewel, A.; de Vries, H.E.; Aronica, E.; Gorter, J.A.

    2012-01-01

    Purpose: Previous studies have shown that inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin prevents epileptogenesis after pharmacologically induced status epilepticus (SE) in rat models of temporal lobe epilepsy. Because rapamycin is also known for its immunosuppressant

  15. Selective Class I HDAC Inhibition Suppresses Hypoxia-Induced Cardiopulmonary Remodeling Through an Anti-Proliferative Mechanism

    Science.gov (United States)

    Cavasin, Maria A.; Demos-Davies, Kim; Horn, Todd R.; Walker, Lori A.; Lemon, Douglas D.; Birdsey, Nicholas; Weiser-Evans, Mary C. M.; Harral, Jules; Irwin, David C.; Anwar, Adil; Yeager, Michael E.; Li, Min; Watson, Peter A.; Nemenoff, Raphael A.; Buttrick, Peter M.; Stenmark, Kurt R.; McKinsey, Timothy A.

    2012-01-01

    Rationale Histone deacetylase (HDAC) inhibitors are efficacious in models of hypertension-induced left ventricular (LV) heart failure. The consequences of HDAC inhibition in the context of pulmonary hypertension (PH) with associated right ventricular (RV) cardiac remodeling are poorly understood. Objective This study was performed to assess the utility of selective small molecule inhibitors of class I HDACs in a pre-clinical model of PH. Methods and Results Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs −1, −2 and −3. The compound reduced pulmonary arterial pressure (PAP) more dramatically than tadalafil, a standard-of-care therapy for human PH that functions as a vasodilator. MGCD0103 improved pulmonary artery (PA) acceleration time (PAAT) and reduced systolic notching of the PA flow envelope, suggesting a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced PAP in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening due to suppression of smooth muscle cell proliferation. RV function was maintained in MGCD0103 treated animals. Although the class I HDAC inhibitor only modestly reduced RV hypertrophy, it had multiple beneficial effects on the RV, which included suppression of pathological gene expression, inhibition of pro-apoptotic caspase activity, and repression of pro-inflammatory protein expression. Conclusions By targeting distinct pathogenic mechanisms, isoform-selective HDAC inhibitors have potential as novel therapeutics for PH that will complement vasodilator standards-of-care. PMID:22282194

  16. Inhibition of akt enhances the chemopreventive effects of topical rapamycin in mouse skin

    Science.gov (United States)

    Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Rusche, Jadrian J.; Petricoin, Emmanuel; Calvert, Valerie; Einspahr, Janine G.; Dickinson, Jesse; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

    2016-01-01

    The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

  17. Rapamycin inhibits IGF-1 stimulated cell motility through PP2A pathway.

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    Lei Liu

    2010-05-01

    Full Text Available Serine/threonine (Ser/Thr protein phosphatase 2A (PP2A has been implicated as a novel component of the mammalian target of rapamycin (mTOR signaling pathway. Recently we have demonstrated that mTOR regulates cell motility in part through p70 S6 kinase 1 (S6K1 and eukaryotic initiation factor 4E (eIF4E binding protein 1 (4E-BP1 pathways. Little is known about the role of PP2A in the mTOR-mediated cell motility. Here we show that rapamycin inhibited the basal or insulin-like growth factor 1 (IGF-1-induced motility of human Ewing sarcoma (Rh1 and rhabdomyosarcoma (Rh30 cells. Treatment of the cells with rapamycin activated PP2A activity, and concurrently inhibited IGF-1 stimulated phosphorylation of Erk1/2. Inhibition of Erk1/2 with PD98059 did not significantly affect the basal mobility of the cells, but dramatically inhibited IGF-1-induced cell motility. Furthermore, inhibition of PP2A with okadaic acid significantly attenuated the inhibitory effect of rapamycin on IGF-1-stimulated phosphorylation of Erk1/2 as well as cell motility. Consistently, expression of dominant negative PP2A conferred resistance to IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. Expression of constitutively active MKK1 also attenuated rapamycin inhibition of IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. The results suggest that rapamycin inhibits cell motility, in part by targeting PP2A-Erk1/2 pathway.

  18. Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia

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    Bai, Hualong; Lee, Jung Seok; Chen, Elizabeth; Wang, Mo; Xing, Ying; Fahmy, Tarek M.; Dardik, Alan

    2017-01-01

    Prosthetic grafts and patches are commonly used in cardiovascular surgery, however neointimal hyperplasia remains a significant concern, especially under low flow conditions. We hypothesized that delivery of rapamycin from nanoparticles (NP) covalently attached to patches allows sustained site-specific delivery of therapeutic agents targeted to inhibit localized neointimal hyperplasia. NP were covalently linked to pericardial patches using EDC/NHS chemistry and could deliver at least 360 ng rapamycin per patch without detectable rapamycin in serum; nanoparticles were detectable in the liver, kidney and spleen but no other sites within 24 hours. In a rat venous patch angioplasty model, control patches developed robust neointimal hyperplasia on the patch luminal surface characterized by Eph-B4-positive endothelium and underlying SMC and infiltrating cells such as macrophages and leukocytes. Patches delivering rapamycin developed less neointimal hyperplasia, less smooth muscle cell proliferation, and had fewer infiltrating cells but retained endothelialization. NP covalently linked to pericardial patches are a novel composite delivery system that allows sustained site-specific delivery of therapeutics; NP delivering rapamycin inhibit patch neointimal hyperplasia. NP linked to patches may represent a next generation of tissue engineered cardiovascular implants.

  19. Novel Pathway for Hypoxia-Induced Proliferation and Migration in Human Mesenchymal Stem Cells: Involvement of HIF-1α, FASN, and mTORC1.

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    Lee, Hyun Jik; Ryu, Jung Min; Jung, Young Hyun; Oh, Sang Yub; Lee, Sei-Jung; Han, Ho Jae

    2015-07-01

    The control of stem cells by oxygen signaling is an important way to improve various stem cell physiological functions and metabolic nutrient alteration. Lipid metabolism alteration via hypoxia is thought to be a key factor in controlling stem cell fate and function. However, the interaction between hypoxia and the metabolic and functional changes to stem cells is incompletely described. This study aimed to identify hypoxia-inducible lipid metabolic enzymes that can regulate umbilical cord blood (UCB)-derived human mesenchymal stem cell (hMSC) proliferation and migration and to demonstrate the signaling pathway that controls functional change in UCB-hMSCs. Our results indicate that hypoxia treatment stimulates UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was controlled by the hypoxia-inducible factor-1 alpha (HIF-1α)/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation as well as UCB-hMSC proliferation and migration. RAPTOR small interfering RNA transfection significantly inhibited hypoxia-induced proliferation and migration. Hypoxia-induced mTOR also regulated CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as that of c-myc, p-cofilin, profilin, and Rho GTPase. Taken together, the results suggest that mTORC1 mainly regulates UCB-hMSC proliferation and migration under hypoxia conditions via control of cell cycle and F-actin organization modulating factors. In conclusion, the HIF-1α/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipid metabolism with proliferation and migration in UCB-hMSCs. Stem Cells 2015;33:2182-2195. © 2015 AlphaMed Press.

  20. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast

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    Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

    2013-01-01

    Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. PMID:23551936

  1. Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells

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    Xiaodong Mu

    2013-01-01

    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2 and vascular endothelial growth factor (VEGF gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.

  2. Inhibition of mammalian target of rapamycin decreases intrarenal oxygen availability and alters glomerular permeability.

    Science.gov (United States)

    Sivertsson, Ebba; Friederich-Persson, Malou; Öberg, Carl M; Fasching, Angelica; Hansell, Peter; Rippe, Bengt; Palm, Fredrik

    2017-09-27

    Increased kidney oxygen consumption causing tissue hypoxia is suggested as a common pathway to chronic kidney disease. Mammalian target of rapamycin (mTOR) regulates cell proliferation and mitochondrial function. mTOR inhibitors, e.g. rapamycin, are used clinically to prevent graft rejection. mTOR has been identified as a key player in diabetes, which has stimulated the use of mTOR inhibitors to counter diabetic nephropathy. However, the effect of mTOR inhibition on kidney oxygen consumption is unknown. We therefore investigated the effects of mTOR inhibition on in vivo kidney function, oxygen homeostasis and glomerular permeability. Control and streptozotocin-induced diabetic rats were chronically treated with rapamycin and the functional consequences studied fourteen days thereafter. In both groups, mTOR inhibition induced mitochondrial uncoupling resulting in increased total kidney oxygen consumption and decreased intrarenal oxygen availability. Concomitantly, mTOR inhibition induced tubular injury, as estimated from urinary excretion of kidney injury molecule-1 (KIM-1), and reduced urinary protein excretion. The latter corresponded to reduced sieving coefficient for large molecules. In conclusion, mTOR inhibition induces mitochondrial dysfunction leading to decreased oxygen availability in normal and diabetic kidneys, which translates to increased KIM-1 in the urine. Reduced proteinuria after mTOR inhibition is an effect of reduced glomerular permeability for large molecules. Since hypoxia has been suggested as a common pathway to development of chronic kidney disease, mTOR inhibition to patients with pre-existing nephropathy should be used with caution since it may accelerate the progression of disease. Copyright © 2017, American Journal of Physiology-Renal Physiology.

  3. PTEN and rapamycin inhibiting the growth of K562 cells through regulating mTOR signaling pathway

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    Chen Hao

    2008-12-01

    Full Text Available Abstract Objective To investigate, in vitro, the regulatory effects of tumor-suppressing gene PTEN on mTOR (mammalian target of rapamycin signaling pathway, the effects of transfected PTEN and rapamycin on the growth inhibition, and apoptosis induction for human leukemia cell line K562 cells. Methods K562 cells were transfected with recombined adenovirus-PTEN vector containing green fluorescent protein (Ad-PTEN-GFP, followed by the treatment of the cells with or without rapamycin. The proliferation inhibition rate and apoptotic rate of these transfected and/or rapamycin treated K562 cells were measured by MTT assay and flow cytometry (FCM, the expression levels of PTEN-, mTOR-, cyclinD1- and P27kip1- mRNA were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR, the protein expression levels of PTEN, Akt, p-Akt were detected by western blotting. Results The proliferation of K562 cells was inhibited by PTEN gene transfection with/without the treatment of rapamycin. The expression levels of PTEN- and P27kip1- mRNA were up-regulated, and the mTOR- and cyclinD1- mRNA were down-regulated in K562 cells after the cells transfected with wild type PTEN gene and treated with rapamycin. Conclusion PTEN and rapamycin inhibited mTOR expression by acting as an upstream regulator of mTOR. Low dose rapamycin in combination with over-expressed PTEN might have synergistic effects on inhibiting the proliferation and promoting apoptosis of K562 cells.

  4. Inhibition of p70S6K does not mimic the enhancement of Akt phosphorylation by rapamycin.

    Science.gov (United States)

    Wang, Xuerong; Yue, Ping; Tao, Hui; Sun, Shi-Yong

    2017-08-01

    It has been suggested that the mTOR complex 1 (mTORC1)/p70S6K axis represses upstream PI3K/Akt signaling through phosphorylation of IRS-1 and its subsequent degradation. One potential and current model that explains Akt activation induced by the mTOR inhibitor rapamycin is the relief of mTORC1/p70S6K-mediated feedback inhibition of IRS-1/PI3K/Akt signaling, although this has not been experimentally proven. In this study, we found that chemical inhibition of p70S6K did not increase Akt phosphorylation. Surprisingly, knockdown of p70S6K even substantially inhibited Akt phosphorylation. Hence, p70S6K inhibition clearly does not mimic the activation of Akt by rapamycin. Inhibition or enforced activation of p70S6K did not affect the ability of rapamycin to increase Akt phosphorylation. Moreover, inhibition of mTORC1 with either rapamycin or raptor knockdown did not elevate IRS-1 levels, despite potently increasing Akt phosphorylation. Critically, knockdown or knockout of IRS-1 or IRS-2 failed to abolish the ability of rapamycin to increase Akt phosphorylation. Therefore, IRS-1 and IRS-2 are not essential for mediating rapamycin-induced Akt activation. Collectively, our findings suggest that Akt activation by rapamycin or mTORC1 inhibition is unlikely due to relief of p70S6K-mediated feedback inhibition of IRS-1/PI3K/Akt signaling.

  5. Inhibiting the Mammalian target of rapamycin blocks the development of experimental cerebral malaria.

    Science.gov (United States)

    Gordon, Emile B; Hart, Geoffrey T; Tran, Tuan M; Waisberg, Michael; Akkaya, Munir; Skinner, Jeff; Zinöcker, Severin; Pena, Mirna; Yazew, Takele; Qi, Chen-Feng; Miller, Louis H; Pierce, Susan K

    2015-06-02

    Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host's immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4(+) and CD8(+) T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection

  6. Molecular characterization and mRNA expression of hypoxia inducible factor-1 and cognate inhibiting factor in Macrobrachium nipponense in response to hypoxia.

    Science.gov (United States)

    Sun, Shengming; Xuan, Fujun; Fu, Hongtuo; Ge, Xianping; Zhu, Jian; Qiao, Hui; Jin, Shubo; Zhang, Wenyi

    2016-01-01

    Hypoxia inducible factors (HIFs) are considered to be the master switches of oxygen-dependent gene expression in mammalian species. Currently, very little is known about the function of this important pathway or the molecular structures of key players in the hypoxia-sensitive Oriental River Prawn Macrobrachium nipponense. In this study, HIFs-1α (HIF-1α), -1β (HIF-1β) and HIF 1-alpha inhibitor (FIH-1) from M. nipponense were cloned. The 4903-bp cDNA of M. nipponense HIF-1α (MnHIF-1α) encodes a protein of 1088 aa, M. nipponense HIF-1β (MnHIF-1β) spans 2042bp encoding 663 aa and the 1163bp M. nipponense FIH-1 (MnFIH-1) specifies a polypeptide of 345 aa. MnHIF-1 and MnFIH-1 homologs exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate isoforms. Phylogenetic analysis identifies that genetic diversification of HIF-1 and FIH-1 occurred within the invertebrate lineage, indicating functional specialization of the oxygen sensing pathways in this group. Quantitative real-time RT-PCR demonstrated that MnHIF-1 and MnFIH-1 mRNA are expressed in different tissues and exhibit transcriptional responses to severe hypoxia in gill and muscle tissue, consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. The role of HIF-1α in response to hypoxia was further investigated in the gills and muscles of prawns using in situ hybridization. These results suggested that HIF-1α plays an important role in oxygen sensing and homeostasis in M. nipponense. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Low-Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

    Science.gov (United States)

    Hester, J; Schiopu, A; Nadig, S N; Wood, K J

    2012-01-01

    Regulatory T cells (Treg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote Treg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human Treg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic Treg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2−/−Il2rg−/− mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4+ but not CD8+ T lymphocytes were sensitive to Treg and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of Treg-based immunosuppressive protocols in clinical practice. By inhibiting TA, Treg and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival PMID:22500984

  8. Sirtuin 6 suppresses hypoxia-induced inflammatory response in human osteoblasts via inhibition of reactive oxygen species production and glycolysis-A therapeutic implication in inflammatory bone resorption.

    Science.gov (United States)

    Hou, Kuo-Liang; Lin, Sze-Kwan; Chao, Ling-Hsiu; Hsiang-Hua Lai, Eddie; Chang, Cheng-Chi; Shun, Chia-Tung; Lu, Wan-Yu; Wang, Jyh-Horng; Hsiao, Michael; Hong, Chi-Yuan; Kok, Sang-Heng

    2017-03-01

    Elevated glycolytic activity and redox imbalance induced by tissue hypoxia are common phenomena of chronic inflammation, including inflammatory bone diseases such as arthritis. However, relation between glycolysis and redox signaling in the inflammatory milieu is unclear. The histone deacetylase sirtuin 6 (SIRT6) is a crucial modulator of inflammation and glucose metabolism, and it is also involved in cellular protection against oxidative injury. The aims of the study were to examine the connection between glycolysis and reactive oxygen species (ROS) production in human osteoblastic cells (HOB) and whether SIRT6 modulates inflammatory response via regulation of glycolytic activity and ROS generation. In HOB cultured under hypoxia, expression of lactate dehydrogenase A (LDHA), lactate production and ROS generation were examined. The reciprocal effects between lactate and ROS production and their impact on inflammatory cytokine induction were assessed. The action of SIRT6 on the above reactions was determined. In a rat model of collagen-induced arthritis (CIA), the relation between inflammatory activity and osteoblastic expression of LDHA, level of oxidative lesions, Cyr61 synthesis and macrophage recruitment were examined in joints with or without lentiviral-SIRT6 gene therapy. Results showed that hypoxia stress enhanced lactate and LDHA production in HOB. ROS generation was also increased, and there was a positive feedback between glycolysis and ROS formation. Overexpression of SIRT6 attenuated hypoxia-enhanced glycolysis and ROS generation. Hypoxia-induced expressions of Cyr61, TNF-α, IL-1β, and IL-6 were suppressed by SIRT6 and the inhibitory effects overlapped with antiglycolytic and antioxidation mechanisms. In the model of CIA, forced expression of SIRT6 ameliorated disease progression, osteoblastic synthesis of Cyr61, and macrophage recruitment. More importantly, expression of LDHA and oxidative lesions were decreased in osteoblasts of SIRT6-treated joints

  9. Combination of rapamycin, CI-1040, and 17-AAG inhibits metastatic capacity of prostate cancer via Slug inhibition.

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    Guanxiong Ding

    Full Text Available Though prostate cancer (PCa has slow progression, the hormone refractory (HRCP and metastatic entities are substantially lethal and lack effective treatments. Transcription factor Slug is critical in regulating metastases of various tumors including PCa. Here we studied targeted therapy against Slug using combination of 3 drugs targeting 3 pathways respectively converging via Slug and further regulating PCa metastasis. Using in vitro assays we confirmed that Slug up-regulation incurred inhibition of E-cadherin that was anti-metastatic, and inhibited Bim-regulated cell apoptosis in PCa. Upstream PTEN/Akt, mTOR, Erk, and AR/Hsp90 pathways were responsible for Slug up-regulation and each of these could be targeted by rapamycin, CI-1040, and 17-AAG respectively. In 4 PCa cell lines with different traits in terms of PTEN loss and androgen sensitivity we tested the efficacy of mono- and combined therapy with the drugs. We found that metastatic capacity of the cells was maximally inhibited only when all 3 drugs were combined, due to the crosstalk between the pathways. 17-AAG decreases Slug expression via blockade of HSP90-dependent AR stability. Combination of rapamycin and CI-1040 diminishes invasiveness more potently in PCa cells that are androgen insensitive and with PTEN loss. Slug inhibited Bim-mediated apoptosis that could be rescued by mTOR/Erk/HSP90 inhibitors. Using mouse models for circulating PCa DNA quantification, we found that combination of mTOR/Erk/HSP90 inhibitors reduced circulating PCa cells in vivo significantly more potently than combination of 2 or monotherapy. Conclusively, combination of mTOR/Erk/Hsp90 inhibits metastatic capacity of prostate cancer via Slug inhibition.

  10. Environmental Enrichment Prevent the Juvenile Hypoxia-Induced Developmental Loss of Parvalbumin-Immunoreactive Cells in the Prefrontal Cortex and Neurobehavioral Alterations Through Inhibition of NADPH Oxidase-2-Derived Oxidative Stress.

    Science.gov (United States)

    Zhang, Mingqiang; Wu, Jing; Huo, Lan; Luo, Liang; Song, Xi; Fan, Fei; Lu, Yiming; Liang, Dong

    2016-12-01

    We compared the expression of phenotype of parvalbumin (PV)-immunoreactive cells in the prefrontal cortex (PFC) of juvenile rats reared in enriched environment (EE) after daily intermittent hypoxia (IH) exposure to those reared in standard environment (SE) and investigated the involvement of NADPH oxidase-2 (NOX2)-derived oxidative stress in the IH-induced neurodevelopmental and neurobehavioral consequences in a juvenile rat model of obstructive sleep apnea. Postnatal day 21 (P21) rats were exposed to IH or room air 8 h daily for 14 consecutive days. After the daily exposure, the rats were raised in SE or EE. In the PFC of P34 rats, we determined the impact (i) of IH exposures on NOX2-derived oxidative stress and PV immunoreactivity, (ii) of pharmacological NOX2 inhibition on IH-induced oxidative stress and PV immunoreactivity, and (iii) of EE on the IH-induced oxidative stress and PV immunoreactivity. Behavioral testing of psychiatric anxiety was carried out consecutively in the open-field test and elevated plus maze at P35 and P36. The results showed IH exposures increased NOX2 expression in the PFC of P34 rats, which was accompanied with elevation of NOX activity and indirect markers of oxidative stress (4-HNE). IH exposures increased 4-HNE immunoreactivity in cortical PV cells, which was accompanied with reduction of PV immunoreactivity. Treatment of IH rats with the antioxidant/NOX inhibitor apocynin prevented the PV cells loss in the PFC and reversed the IH-induced psychiatric anxiety. EE attenuated the NOX2-derived oxidative stress and reversed the PV-immunoreactivity reduction in the PFC induced by IH. Our data suggest that EE might prevent the juvenile hypoxia-induced developmental loss of PV cells in the PFC and attenuate the neurobehavioral alterations through inhibition of NOX2-derived oxidative stress.

  11. Hypoxia-inducible factor 1α participates in hypoxia-induced epithelial-mesenchymal transition via response gene to complement 32.

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    Zhu, Liang; Zhao, Qiu

    2017-08-01

    The aim of the present study was to explore the function of response gene to complement 32 (RGC-32) in hypoxia-induced epithelial-mesenchymal transition (EMT) in pancreatic cancer. Three kinds of hypoxia-inducible factor 1α (HIF-1α) small interfering (si)RNA were synthesized and the different effects on the expression of HIF-1α were detected by western blotting. In human pancreatic cancer BxPC-3 cells, HIF-1α levels were diminished using siRNA transfection or HIF-1α inhibitor pretreatment, and the expression levels of RGC-32 and EMT-associated proteins were analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, the protein levels of epithelial marker, E-cadherin, and mesenchymal marker, vimentin, were determined by western blotting. Results demonstrated that HIF-1α-Homo-488 siRNA and HIF-1α-Homo-1216 siRNA diminished the protein level of HIF-1α. Compared with normoxia, hypoxia induced the levels of HIF-1α, RGC-32, N-cadherin and vimentin, but suppressed the expression of E-cadherin and cytokeratins. The inhibition of HIF-1α by HIF-1α-Homo-1216 siRNA transfection or HIF-1α inhibitor repressed hypoxia-induced HIF-1α, RGC-32, N-cadherin and vimentin, but increased the expression of E-cadherin and cytokeratins. When RGC-32 was knocked down, hypoxia-induced vimentin was suppressed; however, hypoxia-suppressed N-cadherin was released. In conclusion, the present results demonstrated that hypoxia induced the expression of HIF-1α to activate the levels of RGC-32, in turn to regulate the expression EMT-associated proteins for EMT. These findings revealed the function of RGC-32 in hypoxia-induced EMT and may have identified a novel link between HIF-1α and EMT for pancreatic cancer therapy.

  12. Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

    Science.gov (United States)

    Broekaart, Diede W M; van Scheppingen, Jackelien; Geijtenbeek, Karlijne W; Zuidberg, Mark R J; Anink, Jasper J; Baayen, Johannes C; Mühlebner, Angelika; Aronica, Eleonora; Gorter, Jan A; van Vliet, Erwin A

    2017-08-01

    Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. The expression of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1β-induced (immuno)proteasome gene expression could be attenuated by rapamycin. Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy. Wiley Periodicals, Inc. © 2017

  13. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF

    National Research Council Canada - National Science Library

    Koivunen, Peppi; Hirsilä, Maija; Remes, Anne M; Hassinen, Ilmo E; Kivirikko, Kari I; Myllyharju, Johanna

    ... (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH...

  14. Rapamycin inhibits ox-LDL-induced inflammation in human endothelial cells in vitro by inhibiting the mTORC2/PKC/c-Fos pathway.

    Science.gov (United States)

    Sun, Juan-Juan; Yin, Xiao-Wei; Liu, Hui-Hui; Du, Wen-Xiu; Shi, Lu-Yao; Huang, Ya-Bo; Wang, Fen; Liu, Chun-Feng; Cao, Yong-Jun; Zhang, Yan-Lin

    2017-10-26

    Rapamycin and its derivative possess anti-atherosclerosis activity, but its effects on adhesion molecule expression and macrophage adhesion to endothelial cells during atherosclerosis remain unclear. In this study we explored the effects of rapamycin on ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells in vitro and the underlying mechanisms. Ox-LDL (6-48 μg/mL) dose-dependently increased the protein levels of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in human umbilical vein endothelial cells (HUVECs), whereas pretreatment with rapamycin (1-10 μmol/L) dose-dependently inhibited ox-LDL-induced increase in the adhesion molecule expression and macrophage adhesion to endothelial cells. Knockdown of mTOR or rictor, rather than raptor, mimicked the effects of rapamycin. Ox-LDL (100 μg/mL) time-dependently increased PKC phosphorylation in HUVECs, which was abolished by rapamycin or rictor siRNA. Pretreatment with PKC inhibitor staurospo¬rine significantly reduced ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells, whereas pretreatment with PKC activator PMA/TPA attenuated the inhibitory effect of rapamycin on adhesion molecule expression. Ox-LDL (100 μg/mL) time-dependently increased c-Fos levels in HUVECs, and pretreatment with rapamycin or rictor siRNA significantly decreased expression of c-Fos. Knockdown of c-Fos antagonized ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells. Our results demonstrate that rapamycin reduces ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells by inhibiting mTORC2, but not mTORC1, and mTORC2 acts through the PKC/c-Fos signaling pathway.

  15. Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition

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    Chintala Sreenivasulu

    2012-07-01

    Full Text Available Abstract Background Clear cell renal cell carcinoma (ccRCC accounts for more than 80% of the cases of renal cell carcinoma. In ccRCC deactivation of Von-Hippel-Lindau (VHL gene contributes to the constitutive expression of hypoxia inducible factors 1 and 2 alpha (HIF-α, transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1α by Se-Methylselenocysteine (MSC via stabilization of prolyl hydroxylases 2 and 3 (PHDs and a significant therapeutic synergy when combined with chemotherapy. This study was initiated to investigate the expression of PHDs, HIF-α, and VEGF-A in selected solid cancers, the mechanism of HIF-α inhibition by MSC, and to document antitumor activity of MSC against human ccRCC xenografts. Methods Tissue microarrays of primary human cancer specimens (ccRCC, head & neck and colon were utilized to determine the incidence of PHD2/3, HIF-α, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s of HIF-α inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1α positive, 786–0 (HIF-2α positive and VHL wild type head & neck cancer cells FaDu (HIF-1α were utilized. PHD2 and VHL gene specific siRNA knockdown and inhibitors of PHD2 and proteasome were used to determine their role in the degradation of HIF-1α by MSC. Results We have demonstrated that ccRCC cells express low incidence of PHD2 (32%, undetectable PHD3, high incidence of HIF-α (92%, and low incidence of VEGF-A compared to head & neck and colon cancers. This laboratory was the first to identify MSC as a highly effective inhibitor of constitutively expressed HIF-α in ccRCC tumors. MSC did not inhibit HIF-1α protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1α was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-α were associated with

  16. Inhibition of Hypoxia Inducible Factor Alpha and Astrocyte-Elevated Gene-1 Mediates Cryptotanshinone Exerted Antitumor Activity in Hypoxic PC-3 Cells

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    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.

  17. Rapamycin Protects from Type-I Peritoneal Membrane Failure Inhibiting the Angiogenesis, Lymphangiogenesis, and Endo-MT

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    Guadalupe Tirma González-Mateo

    2015-01-01

    Full Text Available Preservation of peritoneal membrane (PM is essential for long-term survival in peritoneal dialysis (PD. Continuous presence of PD fluids (PDF in the peritoneal cavity generates chronic inflammation and promotes changes of the PM, such as fibrosis, angiogenesis, and lymphangiogenesis. Mesothelial-to-mesenchymal transition (MMT and endothelial-to-mesenchymal transition (Endo-MT seem to play a central role in this pathogenesis. We speculated that Rapamycin, a potent immunosuppressor, could be beneficial by regulating blood and lymphatic vessels proliferation. We demonstrate that mice undergoing a combined PD and Rapamycin treatment (PDF + Rapa group presented a reduced PM thickness and lower number of submesothelial blood and lymphatic vessels, as well as decreased MMT and Endo-MT, comparing with their counterparts exposed to PD alone (PDF group. Peritoneal water transport in the PDF + Rapa group remained at control level, whereas PD effluent levels of VEGF, TGF-β, and TNF-α were lower than in the PDF group. Moreover, the treatment of mesothelial cells with Rapamycin in vitro significantly decreased VEGF synthesis and selectively inhibited the VEGF-C and VEGF-D release when compared with control cells. Thus, Rapamycin has a protective effect on PM in PD through an antifibrotic and antiproliferative effect on blood and lymphatic vessels. Moreover, it inhibits Endo-MT and, at least partially, MMT.

  18. Mammalian target of rapamycin inhibition in polycystic kidney disease: From bench to bedside

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    Hyun-Jung Kim

    2012-09-01

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is the most common life-threatening hereditary disease in the USA resulting in chronic kidney disease and the need for dialysis and transplantation. Approximately 85% of cases of ADPKD are caused by a mutation in the Pkd1 gene that encodes polycystin-1, a large membrane receptor. The Pkd1 gene mutation results in abnormal proliferation in tubular epithelial cells, which plays a crucial role in cyst development and/or growth in PKD. Activation of the proliferative mammalian target of rapamycin (mTOR signaling pathway has been demonstrated in polycystic kidneys from rodents and humans. mTOR inhibition with sirolimus or everolimus decreases cysts in most animal models of PKD including Pkd1 and Pkd2 gene deficient orthologous models of human disease. On the basis of animal studies, human studies were undertaken. Two large randomized clinical trials published in the New England Journal of Medicine of everolimus or sirolimus in ADPKD patients were very unimpressive and associated with a high side-effect profile. Possible reasons for the unimpressive nature of the human studies include their short duration, the high drop-out rate, suboptimal dosing, lack of randomization of “fast” and “slow progressors” and the lack of correlation between kidney size and kidney function in ADPKD. The future of mTOR inhibition in ADPKD is discussed.

  19. Rapamycin Induces Heme Oxygenase-1 in Liver but Inhibits Bile Flow Recovery after Ischemia

    NARCIS (Netherlands)

    Kist, Alwine; Wakkie, Joris; Madu, Max; Versteeg, Ruth; ten Berge, Judith; Nikolic, Andrej; Nieuwenhuijs, Vincent B.; Porte, Robert J.; Padbury, Robert T. A.; Barritt, Greg J.

    Background/Aims. Rapamycin, which is employed in the management of patients undergoing liver surgery, induces the synthesis of heme oxygenase-1 (HO-1) in some non-liver cell types. The aim was to investigate whether rapamycin can induce HO-1 expression in the liver, and to test the effects of

  20. OP16, a novel ent-kaurene diterpenoid, potentiates the antitumor effect of rapamycin by inhibiting rapamycin-induced feedback activation of Akt signaling in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Peng, Ke-Zheng; Ke, Yu; Zhao, Qi; Tian, Fei; Liu, Hong-Min; Hou, Guiqin; Lu, Zhaoming

    2017-09-15

    Hyperactivation of mTOR signaling pathway has been viewed as a significant molecular pathogenesis of cancer. However, inhibition of mTOR by rapamycin and its analogs could induce numerous negative feedback loops to attenuate their therapeutic efficacy. As a traditional Chinese herbal medicine, Rabdosia rubescens has been used to treat esophageal squamous cell carcinoma (ESCC) for hundreds of years, and its major effective component is oridonin. Here we reported that OP16, a novel analog of oridonin, showed potent inhibition of cell proliferation and Akt phosphorylation in ESCC cells. The combination of OP16 and rapamycin possesses synergistic anti-proliferative and pro-apoptotic effects both in ESCC cells and ESCC xenografts, and no obvious adverse effect was observed in vivo. Mechanistic analysis revealed that OP16 could inhibit rapamycin-induced Akt activation through the p70S6K-mediated negative feedback loops, and the combination of OP16 and rapamycin was more effective in activating caspase-dependent apoptotic signaling cascade. This study supports the combined use of OP16 with rapamycin as a feasible and effective therapeutic approach for future treatment of ESCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Rapamycin inhibits IGF-1-mediated up-regulation of MDM2 and sensitizes cancer cells to chemotherapy.

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    Wei Du

    Full Text Available The Murine Double Minute 2 (MDM2 protein is a key regulator of cell proliferation and apoptosis that acts primarily by inhibiting the p53 tumor suppressor. Similarly, the PI3-Kinase (PI3K/AKT pathway is critical for growth factor-mediated cell survival. Additionally, it has been reported that AKT can directly phosphorylate and activate MDM2. In this study, we show that IGF-1 up-regulates MDM2 protein levels in a PI3K/AKT-dependent manner. Inhibition of mTOR by rapamycin or expression of a dominant negative eukaryotic initiation factor 4E binding protein 1 (4EBP1 mutant protein, as well as ablation of eukaryotic initiation factor 4E (eIF4E, efficiently abolishes IGF-1-mediated up-regulation of MDM2. In addition, we show that rapamycin effectively inhibits MDM2 expression and sensitizes cancer cells to chemotherapy. Taken together, this study reveals a novel mechanism by which IGF-1 activates MDM2 via the mTOR pathway, and that pharmacologic inhibition of mTOR combined with chemotherapy may be more effective in treatment of a subset of cancers harboring increased MDM2 activation.

  2. Lysyl oxidase mediates hypoxia-induced radioresistance in non-small cell lung cancer A549 cells.

    Science.gov (United States)

    Gong, Chongwen; Gu, Runxia; Jin, Honglin; Sun, Yao; Li, Zhenyu; Chen, Jing; Wu, Gang

    2016-02-01

    Hypoxia-induced radioresistance has been well known as the main obstacle in cancer radiotherapy. Lysyl oxidase (LOX) was previously demonstrated to play an important role in hypoxia-induced biological behaviors, such as metastasis and angiogenesis, through hypoxia-inducible factor-1α (HIF-1α), which is an important contributing factor to radioresistance in tumor cells. However, how LOX plays a role in hypoxia-induced radioresistance has yet to be determined. Here, we found that LOX expression was in accordance with HIF-1α expression, and LOX expression at the mRNA and protein level, and enzymatic activity were remarkably upregulated in the hypoxic A549 cells, compared with normoxic A549 cells. Inhibition of LOX resulted in the reduction of the ability to repair double-stranded breaks (DSBs), promotion of apoptosis, relief of G2/M cycle arrest, and eventually reduction of hypoxia-induced radioresistance in the hypoxic A549 cells. This suggests that LOX may play an important role in hypoxia-induced radioresistance. Together, our results might suggest a novel potential therapeutic target in the management of non-small cell lung cancer (NSCLC). © 2016 by the Society for Experimental Biology and Medicine.

  3. Inhibition of mTOR by Rapamycin Results in Auditory Hair Cell Damage and Decreased Spiral Ganglion Neuron Outgrowth and Neurite Formation In Vitro

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    Katharina Leitmeyer

    2015-01-01

    Full Text Available Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR by blocking the mTOR complex 1 (mTORC1. mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp. are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzed in vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation.

  4. Rapamycin inhibits the growth and muscle-sparing effects of clenbuterol.

    Science.gov (United States)

    Kline, William O; Panaro, Frank J; Yang, Hayung; Bodine, Sue C

    2007-02-01

    Clenbuterol and other beta2-adrenergic agonists are effective at inducing muscle growth and attenuating muscle atrophy through unknown mechanisms. This study tested the hypothesis that clenbuterol-induced growth and muscle sparing is mediated through the activation of Akt and mammalian target of rapamycin (mTOR) signaling pathways. Clenbuterol was administered to normal weight-bearing adult rats to examine the growth-inducing effects and to adult rats undergoing muscle atrophy as the result of hindlimb suspension or denervation to examine the muscle-sparing effects. The pharmacological inhibitor rapamycin was administered in combination with clenbuterol in vivo to determine whether activation of mTOR was involved in mediating the effects of clenbuterol. Clenbuterol administration increased the phosphorylation status of PKB/Akt, S6 kinase 1/p70(s6k), and eukaryotic initiation factor 4E binding protein 1/PHAS-1. Clenbuterol treatment induced growth by 27-41% in normal rats and attenuated muscle loss during hindlimb suspension by 10-20%. Rapamycin treatment resulted in a 37-97% suppression of clenbuterol-induced growth and a 100% reduction of the muscle-sparing effect. In contrast, rapamycin was unable to block the muscle-sparing effects of clenbuterol after denervation. Clenbuterol was also shown to suppress the expression of the MuRF1 and MAFbx transcripts in muscles from normal, denervated, and hindlimb-suspended rats. These results demonstrate that the effects of clenbuterol are mediated, in part, through the activation of Akt and mTOR signaling pathways.

  5. Hypoxia-induced cytotoxic drug resistance in osteosarcoma is independent of HIF-1Alpha.

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    Jennifer Adamski

    Full Text Available Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1. In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target

  6. Synergistic inhibition of human melanoma proliferation by combination treatment with B-Raf inhibitor BAY43-9006 and mTOR inhibitor Rapamycin

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    Slingluff Craig L

    2005-10-01

    Full Text Available Abstract Background Targeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation. Methods We evaluated whether BAY43-9006, an inhibitor of the B-Raf kinase, and rapamycin, an inhibitor of the mTOR kinase, would inhibit serum-stimulated proliferation of human melanoma cell lines, either alone or in combination. Proliferation was measured by quantitating melanoma cell numbers with a luciferase for ATP. Phosphorylation of proteins downstream of targeted kinase(s was assayed by immunoblots. Statistical significance was determined with the Student-T test. Isobologram analysis was performed to distinguish additive versus synergistic effects of combinations of drugs. Results Serum-stimulated proliferation of multiple human melanoma cell lines was inhibited by BAY43-9006 and by rapamycin. Melanoma cells containing the B-Raf mutation V599E were more sensitive than cells with wild-type B-raf to 10 nM doses of both BAY43-9006 and rapamycin. Regardless of B-Raf mutational status, the combination of low dose rapamycin and BAY43-9006 synergistically inhibited melanoma cell proliferation. As expected, rapamycin inhibited the phosphorylation of mTOR substrates, p70S6K and 4EBP1, and BAY43-9006 inhibited phosphorylation of ERK, which is dependent on B-Raf activity. We also observed unexpected rapamycin inhibition of the phosphorylation of ERK, as well as BAY43-9006 inhibition of the phosphorylation of mTOR substrates, p70S6K and 4EBP1. Conclusion There was synergistic inhibition of melanoma cell proliferation by the combination of rapamycin and BAY 43-9006, and unexpected inhibition of two signaling pathways by agents

  7. Longevity, aging and rapamycin

    OpenAIRE

    Ehninger, Dan; Neff, Frauke; Xie, Kan

    2014-01-01

    The federal drug administration (FDA)-approved compound rapamycin was the first pharmacological agent shown to extend maximal lifespan in both genders in a mammalian species. A major question then is whether the drug slows mammalian aging or if it has isolated effects on longevity by suppressing cancers, the main cause of death in many mouse strains. Here, we review what is currently known about the effects that pharmacological or genetic mammalian target of rapamycin (mTOR) inhibition have o...

  8. Grape polyphenols inhibit Akt/mammalian target of rapamycin signaling and potentiate the effects of gefitinib in breast cancer.

    Science.gov (United States)

    Castillo-Pichardo, Linette; Dharmawardhane, Suranganie F

    2012-01-01

    We recently reported that a combination of dietary grape polyphenols resveratrol, quercetin, and catechin (RQC), at low concentrations, was effective at inhibiting metastatic cancer progression. Herein, we investigate the molecular mechanisms of RQC in breast cancer and explore the potential of RQC as a potentiation agent for the epidermal growth factor receptor (EGFR) therapeutic gefitinib. Our in vitro experiments showed RQC induced apoptosis in gefitinib-resistant breast cancer cells via regulation of a myriad of proapoptotic proteins. Because the Akt/mammalian target of rapamycin (mTOR) signaling pathway is often elevated during development of anti-EGFR therapy resistance, the effect of RQC on the mTOR upstream effector Akt and the negative regulator AMP kinase (AMPK) was investigated. RQC was found to reduce Akt activity, induce the activation of AMPK, and inhibit mTOR signaling in breast cancer cells. Combined RQC and gefitinib decreased gefitinib resistant breast cancer cell viability to a greater extent than RQC or gefitinib alone. Moreover, RQC inhibited Akt and mTOR and activated AMPK even in the presence of gefitinib. Our in vivo experiments showed combined RQC and gefitinib was more effective than the individual treatments at inhibiting mammary tumor growth and metastasis in nude mice. Therefore, RQC treatment inhibits breast cancer progression and may potentiate anti-EGFR therapy by inhibition of Akt/mTOR signaling.

  9. HIF-1α-l-PGDS-PPARγ regulates hypoxia-induced ANP secretion in beating rat atria.

    Science.gov (United States)

    Li, Xiang; Zhang, Ying; Zhang, Bo; Liu, Xia; Hong, Lan; Liu, Li-Ping; Wu, Cheng-Zhe; Cui, Xun

    2018-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) and peroxisome proliferator activated receptor γ (PPARγ) play important roles in cardiovascular diseases. Nevertheless, effects of hypoxia-inducible factor 1α (HIF-1α) on L-PGDS and PPARγ protein levels and its role in hypoxia-induced atrial natriuretic peptide (ANP) secretion are unclear. In perfused beating rat atria, we observed that hypoxia significantly increased HIF-1α protein levels and stimulated ANP secretion, while upregulating L-PGDS. Hypoxia-induced ANP secretion was clearly attenuated by HIF-1α antagonist 2-methoxyestradiol, downregulating both HIF-1α and L-PGDS protein levels. It was also attenuated by L-PGDS antagonists, AT-56 and HQL-49, downregulating L-PGDS protein levels. In addition, hypoxia-induced ANP secretion was accompanied by increased PPARγ protein levels and was strongly attenuated by PPARγ antagonist GW9662. Hypoxia-induced increase in atrial PPARγ protein levels were dramatically inhibited by both 2-methoxyestradiol and AT-56. These results indicated that hypoxia promotes ANP secretion, at least in part, by activating HIF-1α-l-PGDS-PPARγ signaling in beating rat atria. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Salidroside exerts protective effects against chronic hypoxia-induced pulmonary arterial hypertension via AMPKα1-dependent pathways.

    Science.gov (United States)

    Chen, Mayun; Cai, Hui; Yu, Chang; Wu, Peiliang; Fu, Yangyang; Xu, Xiaomei; Fan, Rong; Xu, Cunlai; Chen, Yanfan; Wang, Liangxing; Huang, Xiaoying

    2016-01-01

    Salidroside, an active ingredient isolated from Rhodiola rosea, has shown to exert protective effects against chronic hypoxia-induced pulmonary arterial hypertension (PAH). However, the underlying mechanisms were not well known. Based on our recent reports, we predicted the involvement of adenosine monophosphate-activated protein kinase (AMPK) mediated effects in salidroside regulation of PAH. Firstly, to prove the hypothesis, rats were exposed to chronic hypoxia and treated with increasing concentrations of salidroside or a selective AMPK activator-5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) for 4 weeks. After salidroside or AICAR treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary artery remodeling were attenuated. Then the effects of salidroside or AICAR on hypoxia-induced excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs), which contributed to pulmonary arterial remodeling, were investigated. Our results suggested salidroside, as well as AICAR, reversed hypoxia-induced PASMCs proliferation and apoptosis resistance while AMPK inhibitor Compound C enhanced the effects of hypoxia. To reveal the potential cellular mechanisms, activation of AMPKα1 and expression of the genes related to proliferation and apoptosis were analyzed in PASMCs after salidroside treatment under hypoxia conditions. The results demonstrated salidroside as well as AICAR might inhibit chronic hypoxia-induced PASMCs proliferation via AMPKα1-P53-P27/P21 pathway and reverse apoptosis resistance via AMPKα1-P53-Bax/Bcl-2-caspase 9-caspase 3 pathway.

  11. Human intermittent hypoxia-induced respiratory plasticity is not caused by inflammation.

    Science.gov (United States)

    Beaudin, Andrew E; Waltz, Xavier; Pun, Matiram; Wynne-Edwards, Katherine E; Ahmed, Sofia B; Anderson, Todd J; Hanly, Patrick J; Poulin, Marc J

    2015-10-01

    Ventilatory instability, reflected by enhanced acute hypoxic (AHVR) and hypercapnic (AHCVR) ventilatory responses is a fundamental component of obstructive sleep apnoea (OSA) pathogenesis. Intermittent hypoxia-induced inflammation is postulated to promote AHVR enhancement in OSA, although the role of inflammation in intermittent hypoxia-induced respiratory changes in humans has not been examined. Thus, this study assessed the role of inflammation in intermittent hypoxia-induced respiratory plasticity in healthy humans.In a double-blind, placebo-controlled, randomised crossover study design, 12 males were exposed to 6 h of intermittent hypoxia on three occasions. Prior to intermittent hypoxia exposures, participants ingested (for 4  days) either placebo or the nonsteroidal anti-inflammatory drugs indomethacin (nonselective cyclooxygenase (COX) inhibitor) and celecoxib (selective COX-2 inhibitor). Pre- and post-intermittent hypoxia resting ventilation, AHVR, AHCVR and serum concentration of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α were assessed.Pre-intermittent hypoxia resting ventilation, AHVR, AHCVR and TNF-α concentrations were similar across all three conditions (p≥0.093). Intermittent hypoxia increased resting ventilation and the AHVR similarly across all conditions (p=0.827), while the AHCVR was increased (p=0.003) and TNF-α was decreased (p=0.006) with only selective COX-2 inhibition.These findings indicate that inflammation does not contribute to human intermittent hypoxia-induced respiratory plasticity. Moreover, selective COX-2 inhibition augmented the AHCVR following intermittent hypoxia exposure, suggesting that selective COX-2 inhibition could exacerbate OSA severity by increasing ventilatory instability. Copyright ©ERS 2015.

  12. Inhibition of the mammalian target of rapamycin (mTOR in advanced pancreatic cancer: results of two phase II studies

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    Zhang Yujian

    2010-07-01

    Full Text Available Abstract Background The phosphoinositide 3-kinase (PI3K/Akt pathway is constitutively activated in pancreatic cancer and the mammalian target of rapamycin (mTOR kinase is an important mediator for its signaling. Our recent in vitro studies suggest that prolonged exposure of pancreatic cancer cells to mTOR inhibitors can promote insulin receptor substrate-PI3K interactions and paradoxically increase Akt phosphorylation and cyclin D1 expression in pancreatic cancer cells (negative feedback loop. The addition of erlotinib to rapamycin can down-regulate rapamycin-stimulated Akt and results in synergistic antitumor activity with erlotinib in preclinical tumor models. Methods Two studies prospectively enrolled adult patients with advanced pancreatic cancer, Eastern Cooperative Oncology Group performance status 0-1, adequate hematologic, hepatic and renal parameters and measurable disease. In Study A, temsirolimus was administered intravenously at 25 mg weekly. In Study B, everolimus was administered orally at 30 mg weekly and erlotinib was administered at 150 mg daily. The primary endpoint in both studies was overall survival at 6 months. Secondary endpoints included time to progression, progression-free survival, overall survival, response rate, safety and toxicity. Pretreatment tumor biopsies were analyzed by immunofluorescence and laser scanning cytometry for the expression of pmTOR/mTOR, pAkt/Akt, pErk/Erk, pS6, p4EBP-1 and PTEN. Results Five patients enrolled in Study A; Two patients died within a month (rapid disease progression and hemorrhagic stroke, respectively. One patient developed dehydration and another developed asthenia. Sixteen patients enrolled in Study B.: 12 males, all ECOG PS = 1. Median cycles = 1 (range 1-2. Grade 4 toxicity: hyponatremia (n = 1, Grade 3: diarrhea (n = 1, cholangitis (n = 3, hyperglycemia (n = 1, fatigue (n = 1. Grade 2: pneumonia (n = 2, dehydration (n = 2, nausea (n = 2, neutropenia (n = 1, mucositis (n = 2

  13. Mechanism of Metformin-dependent Inhibition of Mammalian Target of Rapamycin (mTOR) and Ras Activity in Pancreatic Cancer

    Science.gov (United States)

    Nair, Vijayalekshmi; Sreevalsan, Sandeep; Basha, Riyaz; Abdelrahim, Maen; Abudayyeh, Ala; Rodrigues Hoffman, Aline; Safe, Stephen

    2014-01-01

    The antidiabetic drug metformin exhibits both chemopreventive and chemotherapeutic activity for multiple cancers including pancreatic cancer; however, the underlying mechanism of action of metformin is unclear. A recent study showed that metformin down-regulated specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells and tumors, and this was accompanied by down-regulation of several pro-oncogenic Sp-regulated genes. Treatment with metformin or down-regulation of Sp TFs by RNAi also inhibits two major pro-oncogenic pathways in pancreatic cancer cells, namely mammalian target of rapamycin (mTOR) signaling and epidermal growth factor (EGFR)-dependent activation of Ras. Metformin and Sp knockdown by RNAi decreased expression of the insulin-like growth factor-1 receptor (IGF-1R), resulting in inhibition of mTOR signaling. Ras activity was also decreased by metformin and Sp knockdown of EGFR, another Sp-regulated gene. Thus, the antineoplastic activities of metformin in pancreatic cancer are due, in part, to down-regulation of Sp TFs and Sp-regulated IGF-1R and EGFR, which in turn results in inhibition of mTOR and Ras signaling, respectively. PMID:25143389

  14. Cryptotanshinone inhibition of mammalian target of rapamycin pathway is dependent on oestrogen receptor alpha in breast cancer.

    Science.gov (United States)

    Pan, Yanhong; Shi, Junfeng; Ni, Wenting; Liu, Yuping; Wang, Siliang; Wang, Xu; Wei, Zhonghong; Wang, Aiyun; Chen, Wenxing; Lu, Yin

    2017-09-01

    Cryptotanshinone (CPT) has been demonstrated to inhibit proliferation and mammalian target of rapamycin (mTOR) pathway in MCF-7 breast cancer cells. However, the same results are unable to be repeated in MDA-MB-231 cells. Given the main difference of oestrogen receptor α (ERα) between two types of breast cancer cells, It is possibly suggested that CPT inhibits mTOR pathway dependent on ERα in breast cancer. CPT could significantly inhibit cell proliferation of ERα-positive cancer cells, whereas ERα-negative cancer cells are insensitive to CPT. The molecular docking results indicated that CPT has a high affinity with ERα, and the oestrogen receptor element luciferase reporter verified CPT distinct anti-oestrogen effect. Furthermore, CPT inhibits mTOR signalling in MCF-7 cells, but not in MDA-MB-231 cells, which is independent on binding to the FKBP12 and disrupting the mTOR complex. Meanwhile, increased expression of phosphorylation AKT and insulin receptor substrate (IRS1) induced by insulin-like growth factor 1 (IGF-1) was antagonized by CPT, but other molecules of IGF-1/AKT/mTOR signalling pathway such as phosphatase and tensin homolog (PTEN) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) were negatively affected. Finally, the MCF-7 cells transfected with shERα for silencing ERα show resistant to CPT, and p-AKT, phosphorylation of p70 S6 kinase 1 (p-S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) were partially recovered, suggesting ERα is required for CPT inhibition of mTOR signalling. Overall, CPT inhibition of mTOR is dependent on ERα in breast cancer and should be a potential anti-oestrogen agent and a natural adjuvant for application in endocrine resistance therapy. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  15. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

    OpenAIRE

    Arriola Apelo, Sebastian I.; Neuman, Joshua C.; Baar, Emma L.; Syed, Faizan A.; Cummings, Nicole E.; Brar, Harpreet K.; Pumper, Cassidy P.; Kimple, Michelle E.; Lamming, Dudley W.

    2015-01-01

    Summary Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA?approved drug rapamycin has been shown to promote lifespan and delay age?related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long?term prophylactic use of rapamycin as a therapy for age?related diseases. While the beneficial effects of rapamycin are larg...

  16. Vitamin C Supplementation Does not Improve Hypoxia-Induced Erythropoiesis

    OpenAIRE

    Martinez-Bello, Vladimir E.; Sanchis-Gomar, Fabian; Martinez-Bello, Daniel; Olaso-Gonzalez, Gloria; Gomez-Cabrera, Mari Carmen; Viña, Jose

    2012-01-01

    Martinez-Bello,Vladimir E., Fabian Sanchis-Gomar, Daniel Martinez-Bello, Gloria Olaso-Gonzalez, Mari Carmen Gomez-Cabrera, and Jose Viña. Vitamin C Supplementation Does Not Improve Hypoxia-Induced Erythropoiesis. High Alt Med Biol 13:269–274, 2012.—Hypoxia induces reactive oxygen species production. Supplements with antioxidant mixtures can compensate for the decline in red cell membrane stability following intermittent hypobaric hypoxia by decreasing protein and lipid oxidation. We aimed to ...

  17. Structural integration in hypoxia-inducible factors

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Dalei; Potluri, Nalini; Lu, Jingping; Kim, Youngchang; Rastinejad, Fraydoon

    2015-08-20

    The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

  18. Cefminox, a Dual Agonist of Prostacyclin Receptor and Peroxisome Proliferator-Activated Receptor-Gamma Identified by Virtual Screening, Has Therapeutic Efficacy against Hypoxia-Induced Pulmonary Hypertension in Rats

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    Jingwen Xia

    2018-02-01

    Full Text Available Prostacyclin receptor (IP and peroxisome proliferator-activated receptor-gamma (PPARγ are both potential targets for treatment of pulmonary arterial hypertension (PAH. Expression of IP and PPARγ decreases in PAH, suggesting that screening of dual agonists of IP and PPARγ might be an efficient method for drug discovery. Virtual screening (VS of potential IP–PPARγ dual-targeting agonists was performed in the ZINC database. Ten of the identified compounds were further screened, and cefminox was found to dramatically inhibit growth of PASMCs with no obvious cytotoxicity. Growth inhibition by cefminox was partially reversed by both the IP antagonist RO113842 and the PPARγ antagonist GW9662. Investigation of the underlying mechanisms of action demonstrated that cefminox inhibits the protein kinase B (Akt/mammalian target of rapamycin (mTOR signaling pathway through up-regulation of the expression of phosphatase and tensin homolog (PTEN, which is inhibited by GW9662, and enhances cyclic adenosine monophosphate (cAMP production in PASMCs (which is inhibited by RO113842. In a rat model of hypoxia-induced pulmonary hypertension, cefminox displayed therapeutic efficacy not inferior to that of the prostacyclin analog iloprost or the PPARγ agonist rosiglitazone. Our results identified cefminox as a dual agonist of IP and PPARγ that significantly inhibits PASMC proliferation by up-regulation of PTEN and cAMP, suggesting that it has potential for treatment of PAH.

  19. The Protective Effect of Salidroside on Hypoxia-Induced Corpus Cavernosum Smooth Muscle Cell Phenotypic Transformation.

    Science.gov (United States)

    Zhang, Xiang; Zhao, Jian-Feng; Zhao, Fan; Yan, Jun-Feng; Yang, Fan; Huang, Xiao-Jun; Chen, Gang; Fu, Hui-Ying; Lv, Bo-Dong

    2017-01-01

    Salidroside, a major active ingredient isolated from Rhodiola rosea, has a long application in Chinese medical history. It has widely demonstrated effects on fatigue, psychological stress, and depression and exhibits potential antihypoxia activity. Emerging evidence shows that hypoxia is an important independent risk factor for erectile dysfunction (ED). The aim of this study was to clarify the effect of salidroside on hypoxia-induced phenotypic transformation of corpus cavernosum smooth muscle cells (CCSMCs). Our results showed that salidroside decreased the hypoxia-induced expression of collagen and content of vimentin, a corpus cavernosum smooth muscle synthetic protein, in vitro. Simultaneously, salidroside increased expression of the CCSMC contractile proteins, α-smooth muscle actin (α-SMA) and desmin. In vivo, similarly, the expressions of collagen and hypoxia-inducible factor-1α were increased in bilateral cavernous neurectomy (BCN) rats while they were decreased in the salidroside group. Among the phenotypic proteins, α-SMA and desmin increased and vimentin decreased after treating BCN rats with salidroside compared with the BCN alone group. Overall, our results demonstrate that salidroside has the ability to oppose hypoxia and can inhibit the CCSMC phenotypic transformation induced by hypoxia. Salidroside may provide a new treatment method for ED.

  20. The Protective Effect of Salidroside on Hypoxia-Induced Corpus Cavernosum Smooth Muscle Cell Phenotypic Transformation

    Directory of Open Access Journals (Sweden)

    Xiang Zhang

    2017-01-01

    Full Text Available Salidroside, a major active ingredient isolated from Rhodiola rosea, has a long application in Chinese medical history. It has widely demonstrated effects on fatigue, psychological stress, and depression and exhibits potential antihypoxia activity. Emerging evidence shows that hypoxia is an important independent risk factor for erectile dysfunction (ED. The aim of this study was to clarify the effect of salidroside on hypoxia-induced phenotypic transformation of corpus cavernosum smooth muscle cells (CCSMCs. Our results showed that salidroside decreased the hypoxia-induced expression of collagen and content of vimentin, a corpus cavernosum smooth muscle synthetic protein, in vitro. Simultaneously, salidroside increased expression of the CCSMC contractile proteins, α-smooth muscle actin (α-SMA and desmin. In vivo, similarly, the expressions of collagen and hypoxia-inducible factor-1α were increased in bilateral cavernous neurectomy (BCN rats while they were decreased in the salidroside group. Among the phenotypic proteins, α-SMA and desmin increased and vimentin decreased after treating BCN rats with salidroside compared with the BCN alone group. Overall, our results demonstrate that salidroside has the ability to oppose hypoxia and can inhibit the CCSMC phenotypic transformation induced by hypoxia. Salidroside may provide a new treatment method for ED.

  1. [Protective effect of salidroside against high altitude hypoxia-induced brain injury in rats].

    Science.gov (United States)

    Dong, Xiaoru; Zhang, Xiangnan; Li, Dan; Li, Bin; Wang, Jiye; Meng, Shanshan; Luo, Wenjing; Zhang, Wenbin

    2015-10-01

    To observe the protective effect of salidroside against brain injury in rats exposed to hypobaric hypoxia, and investigate the molecular mechanism of salidroside in the prevention of hypobaric hypoxia-induced brain injury. Rats were placed in experiment module simulating 6000 m altitude to establish acute hypobaric hypoxia-induced brain injury models. Their respiratory frequency was observed and recorded. Cell apoptosis in the hippocampal dentate gyrus (DG) was detected by TUNEL assay; the expressions of Ras homolog family member A (RhoA), phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated c-Jun N-terminal kinase (p-JNK) were detected by Western blotting. After acute exposure to 6000 m altitude, the respiratory frequency of the rats increased remarkably. The simulation of hypobaric hypoxia induced cell apoptosis in hippocampal DG region, and salidroside intervention inhibited the process of cell apoptosis. The expressions of RhoA, p-ERK, p-JNK decreased after hypobaric hypoxia exposure. Salidroside intervention reversed RhoA expression and raised the levels of p-ERK and p-JNK. Acute exposure to hypobaric hypoxia can induce cell apoptosis in rat hippocampal DG, and salidroside can protect the cells from the exposure-induced apoptosis.

  2. Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

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    Hagen Thilo

    2011-04-01

    Full Text Available Abstract The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein. We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux. This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.

  3. MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin.

    Science.gov (United States)

    Zhang, Zi-Wei; Guo, Rui-Wei; Lv, Jin-Lin; Wang, Xian-Mei; Ye, Jin-Shan; Lu, Ni-Hong; Liang, Xing; Yang, Li-Xia

    2017-04-29

    Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation, migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection

    Directory of Open Access Journals (Sweden)

    Jorge David Rojas Márquez

    2018-02-01

    Full Text Available The causative agent of Chagas’ disease, Trypanosoma cruzi, affects approximately 10 million people living mainly in Latin America, with macrophages being one of the first cellular actors confronting the invasion during T. cruzi infection and their function depending on their proper activation and polarization into distinct M1 and M2 subtypes. Macrophage polarization is thought to be regulated not only by cytokines and growth factors but also by environmental signals. The metabolic checkpoint kinase mammalian target of rapamycin (mTOR-mediated sensing of environmental and metabolic cues influences macrophage polarization in a complex and as of yet incompletely understood manner. Here, we studied the role of the mTOR pathway in macrophages during T. cruzi infection. We demonstrated that the parasite activated mTOR, which was beneficial for its replication since inhibition of mTOR in macrophages by different inhibitors decreased parasite replication. Moreover, in rapamycin pretreated and infected macrophages, we observed a decreased arginase activity and expression, reduced IL-10 and increased interleukin-12 production, compared to control infected macrophages treated with DMSO. Surprisingly, we also found a reduced iNOS activity and expression in these macrophages. Therefore, we investigated possible alternative mechanisms involved in controlling parasite replication in rapamycin pretreated and infected macrophages. Although, cytoplasmic ROS and the enzyme indoleamine 2, 3-dioxygenase (IDO were not involved, we observed a significant increase in IL-6, TNF-α, and IL-1β production. Taking into account that IL-1β is produced by activation of the cytoplasmic receptor NLRP3, which is one of the main components of the inflammasome, we evaluated NLRP3 expression during mTOR inhibition and T. cruzi infection. We observed that rapamycin-pretreated and infected macrophages showed a significant increase in NLRP3 expression and produced higher levels of

  5. Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

    Science.gov (United States)

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Danielski, Lucineia G; Petronilho, Fabricia; Carvalho, André F; Quevedo, João

    2017-09-01

    Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol) or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against

  6. Hypoxia-inducible transcription factor-1α promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway

    Directory of Open Access Journals (Sweden)

    Cao GuiQun

    2006-01-01

    Full Text Available Abstract Background Hypoxia-inducible transcription factor-1α (HIF-1α, which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a "master" gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1α on apoptosis by modulating HIF-1α gene expression in A549 cells through both siRNA knock-down and over-expression. Methods A549 cells were transfected with a HIF-1α siRNA plasmid or a HIF-1α expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG (5 mM. The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1, phosphoglycerate kinase 1(PGK1, and hexokinase 1(HK1, were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. Results Knocking down expression of HIF-1α inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1α accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1α on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1α over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. Conclusion During hypoxia in A549 cells, HIF-1α promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis.

  7. Rapamycin inhibits CaCl2-induced thoracic aortic aneurysm formation in rats through mTOR-mediated suppression of proinflammatory mediators.

    Science.gov (United States)

    Cao, Jiumei; Wu, Qihong; Geng, Liang; Chen, Xiaonan; Shen, Weifeng; Wu, Fang; Chen, Ying

    2017-08-01

    The aim of the present study was to investigate the effect of the mammalian target of rapamycin (mTOR) signaling pathway on thoracic aortic aneurysm (TAA) development. The study used a calcium chloride (CaCl2)‑induced rat TAA model to explore the potential role of mTOR signaling pathway in the disease development. Adult male Sprague‑Dawley rats underwent the periarterial exposure of thoracic aorta to either 0.5 M CaCl2 or normal saline, and a subgroup of CaCl2‑treated rats received rapamycin 1 day prior to surgery. Without pre‑administering rapamycin, significantly enhanced phosphorylation of mTOR and expression of proinflammatory cytokines [i.e., tumor necrosis factor α (TNF‑α), interleukin 6 (IL‑6), and interleukin (IL)‑1β] were observed in the CaCl2‑treated aortic segments 2 days post‑treatment compared with the NaCl‑treated segments. At 2 weeks post‑treatment, hematoxylin and eosin and Verhoeff‑Van Gieson staining revealed aneurysmal alteration and disappearance of normal wavy elastic structures in the aortic segments exposed to CaCl2. In contrast, the CaCl2‑induced TAA formation was inhibited by pre‑administering rapamycin to CaCl2‑treated rats, which demonstrated attenuated mTOR phosphorylation and downregulation of the proinflammatory mediators (i.e., TNF‑α, IL‑6, IL‑1β, matrix metallopeptidases 2 and 9) to the control level. Further in vitro cell culture experiments using aortic smooth muscle cell (SMC) suggested that the inhibition of the mTOR signaling pathway by rapamycin could promote the differentiation of SMCs, as reflected by the reduced expression of S100A4 and osteopontin. The present study indicated that the early enhanced mTOR signaling pathway in the TAA development and mTOR inhibitor rapamycin may inhibit CaCl2‑induced TAA formation.

  8. Inhibition of Mammalian Target of Rapamycin Complex 1 Attenuates Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats.

    Science.gov (United States)

    Kumar, Vikash; Wollner, Clayton; Kurth, Theresa; Bukowy, John D; Cowley, Allen W

    2017-10-01

    The goal of the present study was to explore the protective effects of mTORC1 (mammalian target of rapamycin complex 1) inhibition by rapamycin on salt-induced hypertension and kidney injury in Dahl salt-sensitive (SS) rats. We have previously demonstrated that H 2 O 2 is elevated in the kidneys of SS rats. The present study showed a significant upregulation of renal mTORC1 activity in the SS rats fed a 4.0% NaCl for 3 days. In addition, renal interstitial infusion of H 2 O 2 into salt-resistant Sprague Dawley rats for 3 days was also found to stimulate mTORC1 activity independent of a rise of arterial blood pressure. Together, these data indicate that the salt-induced increases of renal H 2 O 2 in SS rats activated the mTORC1 pathway. Daily administration of rapamycin (IP, 1.5 mg/kg per day) for 21 days reduced salt-induced hypertension from 176.0±9.0 to 153.0±12.0 mm Hg in SS rats but had no effect on blood pressure salt sensitivity in Sprague Dawley treated rats. Compared with vehicle, rapamycin reduced albumin excretion rate in SS rats from 190.0±35.0 to 37.0±5.0 mg/d and reduced the renal infiltration of T lymphocytes (CD3 + ) and macrophages (ED1 + ) in the cortex and medulla. Renal hypertrophy and cell proliferation were also reduced in rapamycin-treated SS rats. We conclude that enhancement of intrarenal H 2 O 2 with a 4.0% NaCl diet stimulates the mTORC1 pathway that is necessary for the full development of the salt-induced hypertension and kidney injury in the SS rat. © 2017 American Heart Association, Inc.

  9. Hypoxia-induced retinopathy model in adult zebrafish

    DEFF Research Database (Denmark)

    Cao, Ziquan; Jensen, Lasse D.; Rouhi, Pegah

    2010-01-01

    Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available....... In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1: EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia......-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs....

  10. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    Directory of Open Access Journals (Sweden)

    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  11. Hypoxia-inducible factor as an angiogenic master switch

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    Takuya eHashimoto

    2015-04-01

    Full Text Available Hypoxia-inducible factors (HIFs regulate the transcription of genes that mediate the response to hypoxia. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia. HIFs have been widely studied in physiological and pathological conditions and have been shown to contribute to the pathogenesis of various vascular diseases. In clinical settings, the HIF pathway has been studied for its role in inhibiting carcinogenesis. HIFs might also play a protective role in the pathology of ischemic diseases. Clinical trials of therapeutic angiogenesis after the administration of a single growth factor have yielded unsatisfactory or controversial results, possibly because the coordinated activity of different HIF-induced factors is necessary to induce mature vessel formation. Thus, manipulation of HIF activity to simultaneously induce a spectrum of angiogenic factors offers a superior strategy for therapeutic angiogenesis. Because HIF-2α plays an essential role in vascular remodeling, manipulation of HIF-2α is a promising approach to the treatment of ischemic diseases caused by arterial obstruction, where insufficient development of collateral vessels impedes effective therapy. eIF3e/INT6 interacts specifically with HIF-2α and induces the proteasome inhibitor-sensitive degradation of HIF-2α, independent of hypoxia and VHL. Treatment with eIF3e/INT6 siRNA stabilizes HIF-2α activity even under normoxic conditions and induces the expression of several angiogenic factors, at levels sufficient to produce functional arteries and veins in vivo. We have demonstrated that administration of eIF3e/INT6 siRNA to ischemic limbs or cold-injured brains reduces ischemic damage in animal models. This review summarizes the current understanding of the relationship between HIFs and vascular diseases. We also discuss novel oxygen-independent regulatory proteins that bind HIF-α and the implications of a new method for therapeutic angiogenesis

  12. Rapamycin inhibits mTOR/p70S6K activation in CA3 region of the hippocampus of the rat and impairs long term memory.

    Science.gov (United States)

    Lana, D; Di Russo, J; Mello, T; Wenk, G L; Giovannini, M G

    2017-01-01

    The present study was aimed at establishing whether the mTOR pathway and its downstream effector p70S6K in CA3 pyramidal neurons are under the modulation of the cholinergic input to trigger the formation of long term memories, similar to what we demonstrated in CA1 hippocampus. We performed in vivo behavioral experiments using the step down inhibitory avoidance test in adult Wistar rats to evaluate memory formation under different conditions. We examined the effects of rapamycin, an inhibitor of mTORC1 formation, scopolamine, a muscarinic receptor antagonist or mecamylamine, a nicotinic receptor antagonist, on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition was conducted 30min after i.c.v. injection of rapamycin. Recall testing was performed 1h, 4h or 24h after acquisition. We found that (1) mTOR and p70S6K activation in CA3 pyramidal neurons were involved in long term memory formation; (2) rapamycin significantly inhibited mTOR and of p70S6K activation at 4h, and long term memory impairment 24h after acquisition; (3) scopolamine impaired short but not long term memory, with an early increase of mTOR/p70S6K activation at 1h followed by stabilization at longer times; (4) mecamylamine and scopolamine co-administration impaired short term memory at 1h and 4h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1h and 4h; (5) mecamylamine and scopolamine treatment did not impair long term memory formation; (6) unexpectedly, rapamycin increased mTORC2 activation in microglial cells. Our results demonstrate that in CA3 pyramidal neurons the mTOR/p70S6K pathway is under the modulation of the cholinergic system and is involved in long-term memory encoding, and are consistent with the hypothesis that the CA3 region of the hippocampus is involved in memory mechanisms based on rapid, one-trial object-place learning and recall. Furthermore, our results are in accordance with previous reports that selective

  13. Hypoxia-inducible factor 1-α in chronic gastrointestinal ischemia

    NARCIS (Netherlands)

    Harki, Jihan; Sana, Aria; van Noord, Désirée; van Diest, Paul J; van der Groep, Petra; Kuipers, Ernst J; Moons, Leon M G; Biermann, Katharina; Tjwa, Eric T T L

    Chronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1α (HIF-1α) is expressed under acute hypoxia. We investigated HIF-1α expression in chronic ischemic and

  14. Hypoxia-inducible factor 1-α in chronic gastrointestinal ischemia

    NARCIS (Netherlands)

    J. Harki (Jihan); A. Sana (Aria); D. van Noord (Désirée); P.J. van Diest (Paul); P. van der Groep (Petra); E.J. Kuipers (Ernst); L.M.G. Moons (Leon); K. Biermann (Katharina); E.T.T.L. Tjwa (Eric)

    2014-01-01

    textabstractChronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1α (HIF-1α) is expressed under acute hypoxia. We investigated HIF-1α expression in chronic

  15. Hypoxia-inducible factor 1-alpha in chronic gastrointestinal ischemia

    NARCIS (Netherlands)

    Harki, J.; Sana, A.; Noord, D. van; Diest, P.J. van; Groep, P. van der; Kuipers, E.J.; Moons, L.M.; Biermann, K.; Tjwa, E.T.

    2015-01-01

    Chronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1alpha (HIF-1alpha) is expressed under acute hypoxia. We investigated HIF-1alpha expression in chronic

  16. Role of hypoxia-inducible factor in diabetic myocardial hypertrophy

    African Journals Online (AJOL)

    elevation of hypoxia inducible factor (HIF), which in turn leads to increases in levels of VEGF and other angiogenic factors. This adaptive response delays progression from pathological cardiac hypertrophy to heart failure. In early cardiac hypertrophy, stability of HIF-1 promotes glycolysis, which improves glucose utilization ...

  17. Screening of hypoxia-inducible genes in sporadic ALS.

    LENUS (Irish Health Repository)

    Cronin, Simon

    2008-10-01

    Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

  18. Resveratrol Inhibits Cancer Cell Metabolism by Down Regulating Pyruvate Kinase M2 via Inhibition of Mammalian Target of Rapamycin

    Science.gov (United States)

    Iqbal, Mohd Askandar; Bamezai, Rameshwar N. K.

    2012-01-01

    Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential. PMID:22574221

  19. Hypobaric intermittent hypoxia attenuates hypoxia-induced depressor response.

    Directory of Open Access Journals (Sweden)

    Fang Cui

    Full Text Available Hypobaric intermittent hypoxia (HIH produces many favorable effects in the cardiovascular system such as anti-hypertensive effect. In this study, we showed that HIH significantly attenuated a depressor response induced by acute hypoxia.Sprague-Dawley rats received HIH in a hypobaric chamber simulating an altitude of 5000 m. The artery blood pressure (ABP, heart rate (HR and renal sympathetic nerve activity (RSNA were recorded in anesthetized control rats and rats received HIH. The baseline ABP, HR and RSNA were not different between HIH and control rats. Acute hypoxia-induced decrease in ABP was significantly attenuated in HIH rat compared with control rats. However, acute hypoxia-induced increases in HR and RSNA were greater in HIH rat than in control rats. After removal of bilateral ascending depressor nerves, acute hypoxia-induced depressor and sympathoexcitatory responses were comparable in control and HIH rats. Furthermore, acute hypoxia-induced depressor and sympathoexcitatory responses did not differ between control and HIH groups after blocking ATP-dependent K(+ channels by glibenclamide. The baroreflex function evaluated by intravenous injection of phenylephrine and sodium nitroprusside was markedly augmented in HIH rats compared with control rats. The pressor and sympathoexcitatory responses evoked by intravenous injection of cyanide potassium were also significantly greater in HIH rats than in control rats.Our findings suggest that HIH suppresses acute hypoxia-induced depressor response through enhancement of baroreflex and chemoreflex function, which involves activation of ATP-dependent K(+ channels. This study provides new information and underlying mechanism on the beneficiary effect of HIH on maintaining cardiovascular homeostasis.

  20. Rhodiola crenulata and Its Bioactive Components, Salidroside and Tyrosol, Reverse the Hypoxia-Induced Reduction of Plasma-Membrane-Associated Na,K-ATPase Expression via Inhibition of ROS-AMPK-PKCξ Pathway

    Directory of Open Access Journals (Sweden)

    Shih-Yu Lee

    2013-01-01

    Full Text Available Exposure to hypoxia leads to impaired pulmonary sodium transport, which is associated with Na,K-ATPase dysfunction in the alveolar epithelium. The present study is designed to examine the effect and mechanism of Rhodiola crenulata extract (RCE and its bioactive components on hypoxia-mediated Na,K-ATPase endocytosis. A549 cells were exposed to hypoxia in the presence or absence of RCE, salidroside, or tyrosol. The generation of intracellular ROS was measured by using the fluorescent probe DCFH-DA, and the endocytosis was determined by measuring the expression level of Na,K-ATPase in the PM fraction. Rats exposed to a hypobaric hypoxia chamber were used to investigate the efficacy and underlying mechanism of RCE in vivo. Our results showed that RCE and its bioactive compounds significantly prevented the hypoxia-mediated endocytosis of Na,K-ATPase via the inhibition of the ROS-AMPK-PKCζ pathway in A549 cells. Furthermore, RCE also showed a comparable preventive effect on the reduction of Na,K-ATPase endocytosis and inhibition of AMPK-PKCξ pathway in the rodent model. Our study is the first to offer substantial evidence to support the efficacy of Rhodiola products against hypoxia-associated Na,K-ATPase endocytosis and clarify the ethnopharmacological relevance of Rhodiola crenulata as a popular folk medicine for high-altitude illness.

  1. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  2. 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.

  3. Apelin Protects Primary Rat Retinal Pericytes from Chemical Hypoxia-Induced Apoptosis

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    Li Chen

    2015-01-01

    Full Text Available Pericytes are a population of cells that participate in normal vessel architecture and regulate permeability. Apelin, as the endogenous ligand of G protein-coupled receptor APJ, participates in a number of physiological and pathological processes. To date, the effect of apelin on pericyte is not clear. Our study aimed to investigate the potential protection mechanisms of apelin, with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence staining revealed that pericytes colocalized with APJ in the fibrovascular membranes dissected from proliferative diabetic retinopathy patients. In the in vitro studies, we first demonstrated that the expression of apelin/APJ was upregulated in pericytes under hypoxia, and apelin increased pericytes proliferation and migration. Moreover, knockdown of apelin in pericyte was achieved via lentivirus-mediated RNA interference. After the inhibition of apelin, pericytes proliferation was inhibited significantly in hypoxia culture condition. Furthermore, exogenous recombinant apelin effectively prevented hypoxia-induced apoptosis through downregulating active-caspase 3 expression and increasing the ratio of B cell lymphoma-2 (Bcl-2/Bcl-2 associated X protein (Bax in pericytes. These results suggest that apelin suppressed hypoxia-induced pericytes injury, which indicated that apelin could be a potential therapeutic target for retinal angiogenic diseases.

  4. Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer

    DEFF Research Database (Denmark)

    Havelund, Birgitte Mayland; Spindler, Karen-Lise Garm; Sørensen, Flemming Brandt

    Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer   Birgitte Mayland Havelund1,4 MD, Karen-Lise Garm Spindler1,4 MD, PhD, Flemming Brandt Sørensen2,4 MD, DMSc, Ivan Brandslund3 MD, DMSc, Anders Jakobsen1,4 MD, DMSc. 1Department of Oncology, 2Pathology and 3Biochemistry, Vejle...... Hospital, Vejle, Denmark 4Institute of Regional Health Services Research, University of Southern Denmark, Odense Denmark Background Prognostic and predictive markers are needed for individualizing the treatment of colorectal cancer. Hypoxia-inducible factor 1α (HIF-1α) is a transcription-inducing factor...... is to investigate the predictive and prognostic value of HIF-1α in colorectal cancer. Materials and Methods The project is divided into 3 substudies: 1. Biological and methodological aspects. The expression of HIF-1α measured by immunohistochemistry in paraffin embedded tissue is related to single nucleotide...

  5. Mechanism of hypoxia-induced NFκB

    Science.gov (United States)

    Melvin, Andrew; Mudie, Sharon

    2011-01-01

    The cellular response to hypoxia relies on the activation of a specific transcriptional program. Although, most of the attention is focused on the transcription factor HIF, other transcription factors are also activated in hypoxia. We have recently described the mechanism for hypoxia induced NFκB. We have demonstrated the crucial dependency on the IKK complex as well as in the upstream IKK kinase TAK1. TAK1 and IKK activation is dependent upon the calcium calmodulin kinase, CaMK2 and requires Ubc13 as the E2 ubiquitin conjugation enzyme. We report a role for XIAP as the possible E3-ubiquitin ligase for this system. Interestingly, hypoxia induced IKK mediated phosphorylation of IκBα, does not lead to degradation. Hypoxia prevents IκBα de-sumoylation of Sumo-2/3 chains on critical lysine residues, normally required for K-48 linked polyubiquitination. Our results define a novel pathway regulating NFκB activation. PMID:21325892

  6. Hypoxia Inducible Factor (HIF Hydroxylases as Regulators of Intestinal Epithelial Barrier FunctionSummary

    Directory of Open Access Journals (Sweden)

    Mario C. Manresa

    2017-05-01

    Full Text Available Human health is dependent on the ability of the body to extract nutrients, fluids, and oxygen from the external environment while at the same time maintaining a state of internal sterility. Therefore, the cell layers that cover the surface areas of the body such as the lung, skin, and gastrointestinal mucosa provide vital semipermeable barriers that allow the transport of essential nutrients, fluid, and waste products, while at the same time keeping the internal compartments free of microbial organisms. These epithelial surfaces are highly specialized and differ in their anatomic structure depending on their location to provide appropriate and effective site-specific barrier function. Given this important role, it is not surprising that significant disease often is associated with alterations in epithelial barrier function. Examples of such diseases include inflammatory bowel disease, chronic obstructive pulmonary disease, and atopic dermatitis. These chronic inflammatory disorders often are characterized by diminished tissue oxygen levels (hypoxia. Hypoxia triggers an adaptive transcriptional response governed by hypoxia-inducible factors (HIFs, which are repressed by a family of oxygen-sensing HIF hydroxylases. Here, we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in inflammatory bowel disease through the promotion of intestinal epithelial barrier function through both HIF-dependent and HIF-independent mechanisms. Keywords: Epithelial Barrier, Inflammatory Bowel Disease, Hypoxia, Hypoxia-Inducible Factor (HIF Hydroxylases

  7. Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle.

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    William Hartman

    Full Text Available Hypoxia effects on pulmonary artery structure and function are key to diseases such as pulmonary hypertension. Recent studies suggest that growth factors called neurotrophins, particularly brain-derived neurotrophic factor (BDNF, can influence lung structure and function, and their role in the pulmonary artery warrants further investigation. In this study, we examined the effect of hypoxia on BDNF in humans, and the influence of hypoxia-enhanced BDNF expression and signaling in human pulmonary artery smooth muscle cells (PASMCs.48h of 1% hypoxia enhanced BDNF and TrkB expression, as well as release of BDNF. In arteries of patients with pulmonary hypertension, BDNF expression and release was higher at baseline. In isolated PASMCs, hypoxia-induced BDNF increased intracellular Ca2+ responses to serotonin: an effect altered by HIF1α inhibition or by neutralization of extracellular BDNF via chimeric TrkB-Fc. Enhanced BDNF/TrkB signaling increased PASMC survival and proliferation, and decreased apoptosis following hypoxia.Enhanced expression and signaling of the BDNF-TrkB system in PASMCs is a potential mechanism by which hypoxia can promote changes in pulmonary artery structure and function. Accordingly, the BDNF-TrkB system could be a key player in the pathogenesis of hypoxia-induced pulmonary vascular diseases, and thus a potential target for therapy.

  8. Chronic Normobaric Hypoxia Induces Pulmonary Hypertension in Rats: Role of NF-κB.

    Science.gov (United States)

    Fan, Junming; Fan, Xiaofang; Li, Yang; Ding, Lu; Zheng, Qingqing; Guo, Jinbin; Xia, Dongmei; Xue, Feng; Wang, Yongyu; Liu, Shufang; Gong, Yongsheng

    2016-03-01

    To investigate whether nuclear factor-kappa B (NF-κB) activation is involved in chronic normobaric hypoxia-induced pulmonary hypertension (PH), rats were treated with saline or an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC, 150 mg/kg, sc, twice daily), and exposed to normoxia or chronic normobaric hypoxia with a fraction of inspired oxygen of ∼0.1 for 14 days. Lung tissue levels of NF-κB activity, and interleukin (IL)-1β, IL-6, and cyclooxygenase-2 mRNAs, were determined, and mean pulmonary arterial pressure, right ventricular hypertrophy, and right heart function were evaluated. Compared to the normoxia exposure group, rats exposed to chronic normobaric hypoxia showed an increased NF-κB activity, measured by increased nuclear translocation of p50 and p65 proteins, an increased inflammatory gene expression in the lungs, elevated mean pulmonary arterial blood pressure and mean right ventricular pressure, right ventricular hypertrophy, as assessed by right ventricle-to-left ventricle plus septum weight ratio, and right heart dysfunction. Treatment of hypoxia-exposed rats with PDTC inhibited NF-κB activity, decreased pulmonary arterial blood pressure and right ventricular pressure, and ameliorated right ventricular hypertrophy and right heart dysfunction. Hypoxia exposure increased protein kinase C activity and promoted pulmonary artery smooth muscle cell proliferation in vitro. Our data suggest that NF-κB activation may contribute to chronic normobaric hypoxia-induced PH.

  9. Hypoxia-Inducible Factor-1 as a Therapeutic Target in Endometrial Cancer Management

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    Laura M. S. Seeber

    2010-01-01

    Full Text Available In the Western world, endometrial cancer (EC is the most common malignant tumor of the female genital tract. Solid tumors like EC outgrow their vasculature resulting in hypoxia. Tumor hypoxia is important because it renders an aggressive phenotype and leads to radio- and chemo-therapy resistance. Hypoxia-inducible factor-1 (HIF-1 plays an essential role in the adaptive cellular response to hypoxia and is associated with poor clinical outcome in EC. Therefore, HIF-1 could be an attractive therapeutic target. Selective HIF-1 inhibitors have not been identified. A number of nonselective inhibitors which target signaling pathways upstream or downstream HIF-1 are known to decrease HIF-1 protein levels. In clinical trials for the treatment of advanced and/or recurrent EC are the topoisomerase I inhibitor Topotecan, mTOR-inhibitor Rapamycin, and angiogenesis inhibitor Bevacizumab. Preliminary data shows encouraging results for these agents. Further work is needed to identify selective HIF-1 inhibitors and to translate these into clinical trials.

  10. Effect of Ruscus extract and hesperidin methylchalcone on hypoxia-induced activation of endothelial cells.

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    Bouaziz, N; Michiels, C; Janssens, D; Berna, N; Eliaers, F; Panconi, E; Remacle, J

    1999-12-01

    Ruscus aculeatus extract and the flavonoid hesperidin methylchalcone (HMC) are drugs used in the treatment of chronic venous insufficiency. In the present study, we investigated their effects on the activation of endothelial cells by hypoxia, a condition which mimics venous blood stasis. We observed that Ruscus extract was able to inhibit the activation of endothelial cells by hypoxia: the decrease in ATP content, the activation of phospholipase A2 as well as the subsequent increase in neutrophil adherence with a maximal protection obtained at 50 microg/ml. HMC was also able to inhibit the hypoxia-induced decrease in ATP content. Furthermore, the effects of Ruscus extract and of HMC on this decrease seem to be additive. The biochemical mechanism evidenced in this work might explain some of the beneficial therapeutic effects of these products in the treatment of chronic venous insufficiency patients.

  11. Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1 Downregulates ELOVL1 Gene Expression and Fatty Acid Synthesis in Goat Fetal Fibroblasts

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    Weipeng Wang

    2015-07-01

    Full Text Available Elongation of very-long-chain fatty acids 1 (ELOVL1 is a ubiquitously expressed gene that belongs to the ELOVL family and regulates the synthesis of very-long-chain fatty acids (VLCFAs and sphingolipids, from yeast to mammals. Mammalian target of rapamycin complex 1 (mTORC1 is a central regulator of cell metabolism and is associated with fatty acids synthesis. In this study, we cloned the cDNA that encodes Cashmere goat (Capra hircus ELOVL1 (GenBank Accession number KF549985 and investigated its expression in 10 tissues. ELOVL1 cDNA was 840 bp, encoding a deduced protein of 279 amino acids, and ELOVL1 mRNA was expressed in a wide range of tissues. Inhibition of mTORC1 by rapamycin decreased ELOVL1 expression and fatty acids synthesis in Cashmere goat fetal fibroblasts. These data show that ELOVL1 expression is regulated by mTORC1 and that mTORC1 has significant function in fatty acids synthesis in Cashmere goat.

  12. A Brief Overview of Nitric Oxide and Reactive Oxygen Species Signaling in Hypoxia-Induced Pulmonary Hypertension.

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    Jaitovich, Ariel; Jourd'heuil, David

    2017-01-01

    Pulmonary hypertension (PH) is characterized by increased vasoconstriction and smooth muscle cell hyperplasia driving pathological vascular remodeling of arterial vessels. In this short review, we discuss the primary source of reactive oxygen species (ROS) and nitric oxide (NO) relevant to PH and the mechanism by which dysregulation of their production contributes to PH. Specifically, hypoxia-induced PH is associated with diminished endothelial nitric oxide synthase (eNOS)-derived NO production and increased production of superoxide (O 2 •- ) through eNOS uncoupling and defective mitochondrial respiration. This drives the inhibition of the NO/soluble guanylate cyclase (sGC) pathway and activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) with consequential dysregulation of the pulmonary vasculature. Therapeutics aimed at increasing NO or cGMP bioavailabilities are amenable to hypoxia disease-induced PH. Similarly, strategies targeting HIF-1α are now considered. Overall, pulmonary hypertension including hypoxia-induced PH offers unique opportunities for the rational development of therapeutics centered on modulating redox signaling.

  13. [Inhibitory effect of salidroside on hypoxia-induced apoptosis of corpus cavernosum smooth muscle cells in rats].

    Science.gov (United States)

    Zhao, Jian-Feng; Fu, Hui-Ying; Yang, Fan; Huang, Xiao-Jun; Chen, Gang; Lü, Bo-Dong

    2014-04-01

    To investigate the effect of salidroside on hypoxia-induced apoptosis of corpus cavernosum smooth muscle cells (CCSMCs) in rats. Rat CCSMCs were cultured in vitro by the enzyme digestion method and identified by immunofluorescent staining of anti-alpha-SMA and anti-Desmin. The non-toxic dose of salidroside was determined by MTT assay. Low-oxygen mixed gas (1% O2, 5% CO2, and 94% N2) was piped into a modular incubator chamber to induce hypoxia. The CCSMCs were divided into a normal, a hypoxia, and a 32 microg/mL salidroside intervention group. The apoptosis of the CCSMCs was detected by flow cytometry and the expression of the caspase-3 protein determined by Western blot. The majority of the CCSMCs were positive for alpha-SMA and Desmin at immunofluorescent staining. Salidroside at salidroside significantly reduced hypoxia-induced early apoptosis of CCSMCs ([13.46% +/- 1.87]%, P Salidroside can reduce the expression of cleaved caspase-3 and inhibit hypoxia-induced apoptosis of CCSMCs in rats.

  14. Rapamycin regulates biochemical metabolites

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    Tucci, Paola; Porta, Giovanni; Agostini, Massimiliano; Antonov, Alexey; Garabadgiu, Alexander Vasilievich; Melino, Gerry; Willis, Anne E

    2013-01-01

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth, and deregulation of this pathway is associated with tumorigenesis. p53, and its less investigated family member p73, have been shown to interact closely with mTOR pathways through the transcriptional regulation of different target genes. To investigate the metabolic changes that occur upon inhibition of the mTOR pathway and the role of p73 in this response p...

  15. Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

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    Adriana M Zimnicka

    Full Text Available Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX, a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2 also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC. In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

  16. Interspecific differences in hypoxia-induced gill remodeling in carp.

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    Dhillon, Rashpal S; Yao, Lili; Matey, Victoria; Chen, Bo-Jian; Zhang, An-Jie; Cao, Zhen-Dong; Fu, Shi-Jian; Brauner, Colin J; Wang, Yuxiang S; Richards, Jeffrey G

    2013-01-01

    The gills of many fish, but in particular those of crucian carp (Carassius carassius) and goldfish (Carassius auratus), are capable of extensive remodeling in response to changes in oxygen (O2), temperature, and exercise. In this study, we investigated the interspecific variation in hypoxia-induced gill modeling and hypoxia tolerance in 10 closely related groups of cyprinids (nine species, with two strains of Cyprinus carpio). There was significant variation in hypoxia tolerance, measured as the O2 tension (P(O2)) at which fish lost equilibrium (LOEcrit), among the 10 groups of carp. In normoxia, there was a significant, phylogenetically independent relationship between mass-specific gill surface area and LOEcrit, with the more hypoxia-tolerant carp having smaller gills than their less hypoxia-tolerant relatives. All groups of carp, except the Chinese bream (Megalobrama pellegrini), increased mass-specific gill surface area in response to 48 h of exposure to hypoxia (0.7 kPa) through reductions in the interlamellar cell mass (ILCM) volume. The magnitude of the hypoxia-induced reduction in the ILCM was negatively correlated with LOEcrit (and thus positively correlated with hypoxia tolerance), independent of phylogeny. The hypoxia-induced changes in gill morphology resulted in reduced variation in mass-specific gill surface area among species and eliminated the relationship between LOEcrit and mass-specific gill surface area. While behavioral responses to hypoxia differed among the carp groups, there were no significant relationships between hypoxia tolerance and the Po2 at which aquatic surface respiration (ASR) was initiated or the total number of ASR events observed during progressive hypoxia. Our results are the first to show that the extent of gill remodeling in cyprinids is associated with hypoxia tolerance in a phylogenetically independent fashion.

  17. Hypoxia Inducible Factor-1α (HIF-1 α and its Role in Tumour Progression to Malignancy

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    Gaurav Mrinal Sharma

    2008-07-01

    Full Text Available Hypoxia is a condition in which an area of the body or a tissue is deprived of sufficient supply of oxygen. The lack of nutrients in a hypoxic tissue generally causes apoptosis but some cells are able to adapt to this hypoxic environment and resist apoptosis. This adaptation occurs as a result of gene activation. Hypoxia is a characteristic feature of many cancers and is the stimulus for overexpression of HIF-1α - a basic loop-helix PAS protein family subunit of HIF, which allows the cell to adapt and survive in hostile environment. The presence of hypoxia and HIF-1α is correlated with an increased risk of metastasis and techniques that can inhibit hypoxia inducible factor may be instrumental in finding a cure for cancer.

  18. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Do, Ji Yeon; Choi, Young Keun [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Kook, Hyun [Department of Pharmacology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, Brain Science & Engineering Institute, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Lee, In-Kyu [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Division of Endocrinology and Metabolism, Department of Internal Medicine, Research Institute of Aging and Metabolism, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Park, Dong Ho, E-mail: sarasate2222@gmail.com [Department of Ophthalmology, Kyungpook National University School of Medicine, Daegu (Korea, Republic of)

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O{sub 2}). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice.

  19. Hypoxia-Induced Oxidative Stress Modulation with Physical Activity

    Science.gov (United States)

    Debevec, Tadej; Millet, Grégoire P.; Pialoux, Vincent

    2017-01-01

    Increased oxidative stress, defined as an imbalance between prooxidants and antioxidants, resulting in molecular damage and disruption of redox signaling, is associated with numerous pathophysiological processes and known to exacerbate chronic diseases. Prolonged systemic hypoxia, induced either by exposure to terrestrial altitude or a reduction in ambient O2 availability is known to elicit oxidative stress and thereby alter redox balance in healthy humans. The redox balance modulation is also highly dependent on the level of physical activity. For example, both high-intensity exercise and inactivity, representing the two ends of the physical activity spectrum, are known to promote oxidative stress. Numerous to-date studies indicate that hypoxia and exercise can exert additive influence upon redox balance alterations. However, recent evidence suggests that moderate physical activity can attenuate altitude/hypoxia-induced oxidative stress during long-term hypoxic exposure. The purpose of this review is to summarize recent findings on hypoxia-related oxidative stress modulation by different activity levels during prolonged hypoxic exposures and examine the potential mechanisms underlying the observed redox balance changes. The paper also explores the applicability of moderate activity as a strategy for attenuating hypoxia-related oxidative stress. Moreover, the potential of such moderate intensity activities used to counteract inactivity-related oxidative stress, often encountered in pathological, elderly and obese populations is also discussed. Finally, future research directions for investigating interactive effects of altitude/hypoxia and exercise on oxidative stress are proposed. PMID:28243207

  20. Hypoxia-Induced Oxidative Stress Modulation with Physical Activity.

    Science.gov (United States)

    Debevec, Tadej; Millet, Grégoire P; Pialoux, Vincent

    2017-01-01

    Increased oxidative stress, defined as an imbalance between prooxidants and antioxidants, resulting in molecular damage and disruption of redox signaling, is associated with numerous pathophysiological processes and known to exacerbate chronic diseases. Prolonged systemic hypoxia, induced either by exposure to terrestrial altitude or a reduction in ambient O2 availability is known to elicit oxidative stress and thereby alter redox balance in healthy humans. The redox balance modulation is also highly dependent on the level of physical activity. For example, both high-intensity exercise and inactivity, representing the two ends of the physical activity spectrum, are known to promote oxidative stress. Numerous to-date studies indicate that hypoxia and exercise can exert additive influence upon redox balance alterations. However, recent evidence suggests that moderate physical activity can attenuate altitude/hypoxia-induced oxidative stress during long-term hypoxic exposure. The purpose of this review is to summarize recent findings on hypoxia-related oxidative stress modulation by different activity levels during prolonged hypoxic exposures and examine the potential mechanisms underlying the observed redox balance changes. The paper also explores the applicability of moderate activity as a strategy for attenuating hypoxia-related oxidative stress. Moreover, the potential of such moderate intensity activities used to counteract inactivity-related oxidative stress, often encountered in pathological, elderly and obese populations is also discussed. Finally, future research directions for investigating interactive effects of altitude/hypoxia and exercise on oxidative stress are proposed.

  1. Mechanism of metformin-dependent inhibition of mammalian target of rapamycin (mTOR) and Ras activity in pancreatic cancer: role of specificity protein (Sp) transcription factors.

    Science.gov (United States)

    Nair, Vijayalekshmi; Sreevalsan, Sandeep; Basha, Riyaz; Abdelrahim, Maen; Abudayyeh, Ala; Rodrigues Hoffman, Aline; Safe, Stephen

    2014-10-03

    The antidiabetic drug metformin exhibits both chemopreventive and chemotherapeutic activity for multiple cancers including pancreatic cancer; however, the underlying mechanism of action of metformin is unclear. A recent study showed that metformin down-regulated specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells and tumors, and this was accompanied by down-regulation of several pro-oncogenic Sp-regulated genes. Treatment with metformin or down-regulation of Sp TFs by RNAi also inhibits two major pro-oncogenic pathways in pancreatic cancer cells, namely mammalian target of rapamycin (mTOR) signaling and epidermal growth factor (EGFR)-dependent activation of Ras. Metformin and Sp knockdown by RNAi decreased expression of the insulin-like growth factor-1 receptor (IGF-1R), resulting in inhibition of mTOR signaling. Ras activity was also decreased by metformin and Sp knockdown of EGFR, another Sp-regulated gene. Thus, the antineoplastic activities of metformin in pancreatic cancer are due, in part, to down-regulation of Sp TFs and Sp-regulated IGF-1R and EGFR, which in turn results in inhibition of mTOR and Ras signaling, respectively. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. CX-5461 induces autophagy and inhibits tumor growth via mammalian target of rapamycin-related signaling pathways in osteosarcoma

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    Li L

    2016-09-01

    Full Text Available Leiming Li,1,* Yan Li,2,* Jiansong Zhao,2 Shuli Fan,3 Liguo Wang,1 Xu Li1 1Department of Joint Surgery and Sports Medicine, The First Affiliated Hospital, 2Department of Spine and Joint Surgery, Sheng Jing Hospital, 3Department of Geriatrics, The First Affiliated Hospital, China Medical University, Shenyang, People’s Republic of China *These authors contributed equally to this work Abstract: Osteosarcoma (OS is the most common primary bone tumor, but molecular mechanisms of the disease have not been well understood, and treatment of metastatic OS remains a challenge. Rapid ribosomal RNA synthesis in cancer is transcribed by RNA polymerase I, which results in unbridled cell growth. The recent discovery of CX-5461, a selective RNA polymerase I inhibitor, exerted its inhibitory effect of ribosomal RNA synthesis and antiproliferative potency. Here, we demonstrate that CX-5461 induces G2 arrest in the cell cycle and expression of microtubule-associated protein 1 light chain 3 II isoform in OS cell lines. Autophagic vacuoles could be observed in electron microscopy and 3-methyladenine prevented cell death mediated by CX-5461. Moreover, it significantly augmented phosphorylated AMP-Activated Protein Kinases α (p-AMPK α. (Thr172 expression in U2-OS cells and decreased p-Akt (Ser473 expression in MNNG cells, respectively, which repressed their downstream effector, mammalian target of rapamycin. On the other hand, CX-5461 increased p53 accumulation and messenger RNA level of its target genes, p21, MDM2, and Sestrin1/2 in U2-OS cells. Knockdown of p53 expression markedly impaired cell death as well as the expression of light chain 3-II and p21 induced by CX-5461. It also significantly enhanced doxorubicin-mediated cytotoxic effect in vitro and in vivo together with additive expression of p53, p21, and light chain 3-II in U2-OS cells. Our data indicate that CX-5461 might induce autophagy via mammalian target of rapamycin-associated signaling pathways

  3. Rapamycin Attenuated Cardiac Hypertrophy Induced by Isoproterenol and Maintained Energy Homeostasis via Inhibiting NF-κB Activation

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    Xi Chen

    2014-01-01

    Full Text Available Rapamycin, also known as sirolimus, is an immunosuppressant drug used to prevent rejection organ (especially kidney transplantation. However, little is known about the role of Rapa in cardiac hypertrophy induced by isoproterenol and its underlying mechanism. In this study, Rapa was administrated intraperitoneally for one week after the rat model of cardiac hypertrophy induced by isoproterenol established. Rapa was demonstrated to attenuate isoproterenol-induced cardiac hypertrophy, maintain the structure integrity and functional performance of mitochondria, and upregulate genes related to fatty acid metabolism in hypertrophied hearts. To further study the implication of NF-κB in the protective role of Rapa, cardiomyocytes were pretreated with TNF-α or transfected with siRNA against NF-κB/p65 subunit. It was revealed that the upregulation of extracellular circulating proinflammatory cytokines induced by isoproterenol was able to be reversed by Rapa, which was dependent on NF-κB pathway. Furthermore, the regression of cardiac hypertrophy and maintaining energy homeostasis by Rapa in cardiomyocytes may be attributed to the inactivation of NF-κB. Our results shed new light on mechanisms underlying the protective role of Rapa against cardiac hypertrophy induced by isoproterenol, suggesting that blocking proinflammatory response by Rapa might contribute to the maintenance of energy homeostasis during the progression of cardiac hypertrophy.

  4. The role of endogenous nitric oxide and platelet-activating factor in hypoxia-induced intestinal injury in rats.

    Science.gov (United States)

    Caplan, M S; Hedlund, E; Hill, N; MacKendrick, W

    1994-02-01

    Nitric oxide is an endothelium-derived relaxing factor that promotes capillary integrity, inhibits leukocyte adherence and activation, and scavenges oxygen radicals. Because these effects are important in experimental intestinal injury, we studied the role of NO inhibition on hypoxia-induced bowel necrosis in the rat and investigated the interaction between platelet-activating factor (PAF) and NO in this model. Sprague-Dawley rats were treated with either hypoxia, NO synthase inhibition (NG-methyl-L-arginine [LNMA] or NG-nitro-L-arginine methyl ester [L-NAME]), hypoxia+LNMA, hypoxia+LNMA+NO donors, or hypoxia+LNMA+PAF receptor inhibition. Evaluations included blood pressure, superior mesenteric artery blood flow, arterial blood gases, histological intestinal injury, intestinal myeloperoxidase activity, and intestinal PAF activity. We found that hypoxia alone for 90 minutes (10% O2, partial O2 pressure = 45 mm Hg) or LNMA alone had no detrimental effects. However, hypoxia+LNMA together caused hypotension, metabolic acidosis, intestinal injury, increased intestinal myeloperoxidase activity, and elevated intestinal PAF concentrations that were prevented by exogenous L-arginine. Furthermore, the hypotension and intestinal injury was prevented by PAF receptor blockade. We conclude that endogenous NO protects the intestine from hypoxia-induced inflammation and injury, and the balance between local PAF and NO modulates the outcome of hypoxia-stressed intestine.

  5. Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer

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    Lv Y

    2015-07-01

    Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1 were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

  6. Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish.

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    Hiroyasu Kamei

    2008-08-01

    Full Text Available Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1. IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.

  7. Identification of functional hypoxia inducible factor response elements in the human lysyl oxidase gene promoter.

    Science.gov (United States)

    Wang, Victoria; Davis, David A; Yarchoan, Robert

    2017-08-19

    Human lysyl oxidase (LOX) is a hypoxia-responsive gene whose product catalyzes collagen crosslinking and is thought to be important in cancer metastasis and osteoarthritis. We previously demonstrated that LOX was upregulated by hypoxia inducible factor 2 (HIF-2) more strongly than hypoxia inducible 1 (HIF-1). Here, we further investigated the response of the LOX gene and LOX promoter to HIFs. LOX mRNA, measured by real time reverse transcriptase-PCR, was strongly up-regulated (almost 40-fold), by transfection of HEK-293T cells with a plasmid encoding the HIF-2α subunit of HIF-2, but only three-fold by a plasmid encoding HIF-1α. LOX protein was detectable by Western blot of cells transfected with HIF-2α, but not with HIF-1α. Analysis of a 1487 bp promoter sequence upstream of the human LOX gene revealed 9 potential hypoxia response elements (HREs). Promoter truncation allowed the mapping of two previously unidentified functional HREs, called here HRE8 and HRE7; -455 to -451 and -382 to -386 bp, respectively, upstream of the start codon for LOX. Removal or mutation of these HREs led to a substantial reduction in both HIF-1α and HIF-2α responsiveness. Also, expression of LOX was significantly inhibited by a small molecule specific HIF-2 inhibitor. In conclusion, LOX is highly responsive to HIF-2α and this is largely mediated by two previously unidentified HREs. These observations enhance our understanding of the regulation of this important gene involved in cancer and osteoarthritis, and suggest that these conditions may be targeted by HIF-2 inhibitors. Published by Elsevier Inc.

  8. Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes.

    Science.gov (United States)

    Laukka, Tuomas; Mariani, Christopher J; Ihantola, Tuukka; Cao, John Z; Hokkanen, Juho; Kaelin, William G; Godley, Lucy A; Koivunen, Peppi

    2016-02-19

    The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe(2+) and 2-oxoglutarate-binding residues increased the Km values for these factors 30-80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400-500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes*

    Science.gov (United States)

    Laukka, Tuomas; Mariani, Christopher J.; Ihantola, Tuukka; Cao, John Z.; Hokkanen, Juho; Kaelin, William G.; Godley, Lucy A.; Koivunen, Peppi

    2016-01-01

    The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1–3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe2+ and 2-oxoglutarate-binding residues increased the Km values for these factors 30–80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400–500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization. PMID:26703470

  10. Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

    Science.gov (United States)

    Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

    2017-01-01

    Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

  11. 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.

  12. Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling.

    Science.gov (United States)

    Zhang, Qiuxia; Na, Quan; Song, Weiwei

    2017-10-01

    Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling. © 2017 Japan Society of Obstetrics and Gynecology.

  13. mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice.

    Science.gov (United States)

    Jiao, Yang; Li, Guangxin; Li, Qingle; Ali, Rahmat; Qin, Lingfeng; Li, Wei; Qyang, Yibing; Greif, Daniel M; Geirsson, Arnar; Humphrey, Jay D; Tellides, George

    2017-09-01

    Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln-null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term. Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-β-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective. In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy. © 2017 American Heart Association, Inc.

  14. Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer

    DEFF Research Database (Denmark)

    Havelund, Birgitte Mayland; Spindler, Karen-Lise Garm; Sørensen, Flemming Brandt

    2010-01-01

    Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer  Birgitte Mayland Havelund1,4 MD, Karen-Lise Garm Spindler1,4 MD, PhD, Flemming Brandt Sørensen2,4 MD, DMSc, Ivan Brandslund3 MD, DMSc, Anders Jakobsen1,4 MD, DMSc.1Department of Oncology, 2Pathology and 3Biochemistry, Vejle...... activates transcription of numerous genes associated with angiogenesis, ATP-metabolism, cell-proliferation, glycolysis and apoptosis. HIF-1α is over expressed in many malignant tumors and is reported to play an important role in tumor invasion and progression. The aim of this Ph.D. project is to investigate...... with locally advanced rectal cancer, treated with preoperative chemoradiation (CRT).Preliminary ResultsExpression of HIF-1α has been investigated in diagnostic biopsies from 58 rectal tumors who received preoperative long-course CRT. An association was found between major response to CRT as measured by tumor...

  15. Hypoxia-induced metastasis model in embryonic zebrafish

    DEFF Research Database (Denmark)

    Rouhi, Pegah; Jensen, Lasse D.; Cao, Ziquan

    2010-01-01

    Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring...... of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent Di......I-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average...

  16. Hypoxia-inducible factor 1 and breast cancer metastasis.

    Science.gov (United States)

    Liu, Zhao-Ji; Semenza, Gregg L; Zhang, Hua-Feng

    2015-01-01

    Accumulating evidence has shown that the hypoxic microenvironment, which is critical during cancer development, plays a key role in regulating breast cancer progression and metastasis. The effects of hypoxia-inducible factor 1 (HIF-1), a master regulator of the hypoxic response, have been extensively studied during these processes. In this review, we focus on the roles of HIF-1 in regulating breast cancer cell metastasis, specifically its effects on multiple key steps of metastasis, such as epithelial-mesenchymal transition (EMT), invasion, extravasation, and metastatic niche formation. We also discuss the roles of HIF-1-regulated non-coding RNAs in breast cancer metastasis, and therapeutic opportunities for breast cancer through targeting the HIF-1 pathway.

  17. Role of hypoxia-inducible factors in acute kidney injury.

    Science.gov (United States)

    Andringa, Kelly K; Agarwal, Anupam

    2014-01-01

    Oxygen is vital to mammalian survival. Oxygen deprivation, defined as hypoxia, elicits adaptive responses in cells and tissues, a process regulated by proteins known as hypoxia-inducible factors (HIF). Animal studies have provided compelling data to demonstrate a pivotal role for the HIF pathway in the pathogenesis of acute kidney injury (AKI) that have led to initial human clinical trials examining this pathway in ischemia-reperfusion injury in various organ systems, including the kidney. HIF are master regulators and mediate adaptive responses to low oxygen in tissues and cells. This review will summarize recent key advances in the field highlighting preclinical and clinical studies relevant to the HIF pathway in the pathophysiology of AKI. 2014 S. Karger AG, Basel.

  18. RIG-I Resists Hypoxia-Induced Immunosuppression and Dedifferentiation.

    Science.gov (United States)

    Engel, Christina; Brügmann, Grethe; Lambing, Silke; Mühlenbeck, Larissa H; Marx, Samira; Hagen, Christian; Horváth, Dorottya; Goldeck, Marion; Ludwig, Janos; Herzner, Anna-Maria; Drijfhout, Jan W; Wenzel, Daniela; Coch, Christoph; Tüting, Thomas; Schlee, Martin; Hornung, Veit; Hartmann, Gunther; Van den Boorn, Jasper G

    2017-06-01

    A hypoxic tumor microenvironment is linked to poor prognosis. It promotes tumor cell dedifferentiation and metastasis and desensitizes tumor cells to type-I IFN, chemotherapy, and irradiation. The cytoplasmic immunoreceptor retinoic acid-inducible gene-I (RIG-I) is ubiquitously expressed in tumor cells and upon activation by 5'-triphosphate RNA (3pRNA) drives the induction of type I IFN and immunogenic cell death. Here, we analyzed the impact of hypoxia on the expression of RIG-I in various human and murine tumor and nonmalignant cell types and further investigated its function in hypoxic murine melanoma. 3pRNA-inducible RIG-I-expression was reduced in hypoxic melanoma cells compared with normoxic controls, a phenomenon that depended on the hypoxia-associated transcription factor HIF1α. Still, RIG-I functionality was conserved in hypoxic melanoma cells, whereas responsiveness to recombinant type-I IFN was abolished, due to hypoxia-induced loss of type I IFN receptor expression. Likewise, RIG-I activation in hypoxic melanoma cells, but not exposure to recombinant IFNα, provoked melanocyte antigen-specific CD8+ T-cell and NK-cell attack. Scavenging of hypoxia-induced reactive oxygen species by vitamin C restored the inducible expression of RIG-I under hypoxia in vitro, boosted in vitro anti-melanoma NK- and CD8+ T-cell attack, and augmented 3pRNA antitumor efficacy in vivo These results demonstrate that RIG-I remains operational under hypoxia and that RIG-I function is largely insensitive to lower cell surface expression of the IFNα receptor. RIG-I function could be fortified under hypoxia by the combined use of 3pRNA with antioxidants. Cancer Immunol Res; 5(6); 455-67. ©2017 AACR. ©2017 American Association for Cancer Research.

  19. Functional pathway mapping analysis for hypoxia-inducible factors.

    Science.gov (United States)

    Chuang, Chia-Sheng; Pai, Tun-Wen; Hu, Chin-Hua; Tzou, Wen-Shyong; Dah-Tsyr Chang, Margaret; Chang, Hao-Teng; Chen, Chih-Chia

    2011-06-20

    Hypoxia-inducible factors (HIFs) are transcription factors that play a crucial role in response to hypoxic stress in living organisms. The HIF pathway is activated by changes in cellular oxygen levels and has significant impacts on the regulation of gene expression patterns in cancer cells. Identifying functional conservation across species and discovering conserved regulatory motifs can facilitate the selection of reference species for empirical tests. This paper describes a cross-species functional pathway mapping strategy based on evidence of homologous relationships that employs matrix-based searching techniques for identifying transcription factor-binding sites on all retrieved HIF target genes. HIF-related orthologous and paralogous genes were mapped onto the conserved pathways to indicate functional conservation across species. Quantitatively measured HIF pathways are depicted in order to illustrate the extent of functional conservation. The results show that in spite of the evolutionary process of speciation, distantly related species may exhibit functional conservation owing to conservative pathways. The novel terms OrthRate and ParaRate are proposed to quantitatively indicate the flexibility of a homologous pathway and reveal the alternative regulation of functional genes. The developed functional pathway mapping strategy provides a bioinformatics approach for constructing biological pathways by highlighting the homologous relationships between various model species. The mapped HIF pathways were quantitatively illustrated and evaluated by statistically analyzing their conserved transcription factor-binding elements. hypoxia-inducible factor (HIF), hypoxia-response element (HRE), transcription factor (TF), transcription factor binding site (TFBS), KEGG (Kyoto Encyclopedia of Genes and Genomes), cross-species comparison, orthology, paralogy, functional pathway.

  20. Gene Therapy by Targeted Adenovirus-mediated Knockdown of Pulmonary Endothelial Tph1 Attenuates Hypoxia-induced Pulmonary Hypertension

    Science.gov (United States)

    Morecroft, Ian; White, Katie; Caruso, Paola; Nilsen, Margaret; Loughlin, Lynn; Alba, Raul; Reynolds, Paul N; Danilov, Sergei M; Baker, Andrew H; MacLean, Margaret R

    2012-01-01

    Serotonin is produced by pulmonary arterial endothelial cells (PAEC) via tryptophan hydroxylase-1 (Tph1). Pathologically, serotonin acts on underlying pulmonary arterial cells, contributing to vascular remodeling associated with pulmonary arterial hypertension (PAH). The effects of hypoxia on PAEC-Tph1 activity are unknown. We investigated the potential of a gene therapy approach to PAH using selective inhibition of PAEC-Tph1 in vivo in a hypoxic model of PAH. We exposed cultured bovine pulmonary arterial smooth muscle cells (bPASMCs) to conditioned media from human PAECs (hPAECs) before and after hypoxic exposure. Serotonin levels were increased in hypoxic PAEC media. Conditioned media evoked bPASMC proliferation, which was greater with hypoxic PAEC media, via a serotonin-dependent mechanism. In vivo, adenoviral vectors targeted to PAECs (utilizing bispecific antibody to angiotensin-converting enzyme (ACE) as the selective targeting system) were used to deliver small hairpin Tph1 RNA sequences in rats. Hypoxic rats developed PAH and increased lung Tph1. PAEC-Tph1 expression and development of PAH were attenuated by our PAEC-Tph1 gene knockdown strategy. These results demonstrate that hypoxia induces Tph1 activity and selective knockdown of PAEC-Tph1 attenuates hypoxia-induced PAH in rats. Further investigation of pulmonary endothelial-specific Tph1 inhibition via gene interventions is warranted. PMID:22525513

  1. Hypoxia, Hypoxia-inducible Transcription Factors, and Renal Cancer.

    Science.gov (United States)

    Schödel, Johannes; Grampp, Steffen; Maher, Eamonn R; Moch, Holger; Ratcliffe, Peter J; Russo, Paul; Mole, David R

    2016-04-01

    Renal cancer is a common urologic malignancy, and therapeutic options for metastatic disease are limited. Most clear cell renal cell carcinomas (ccRCC) are associated with loss of von Hippel-Lindau tumor suppressor (pVHL) function and deregulation of hypoxia pathways. This review summarizes recent evidence from genetic and biological studies showing that hypoxia and hypoxia-related pathways play critical roles in the development and progress of renal cancer. We used a systematic search for articles using the keywords hypoxia, HIF, renal cancer, and VHL. Identification of the tumor suppressor pVHL has allowed the characterization of important ccRCC-associated pathways. pVHL targets α-subunits of hypoxia-inducible transcription factors (HIF) for proteasomal degradation. The two main HIF-α isoforms have opposing effects on RCC biology, possibly through distinct interactions with additional oncogenes. Furthermore, HIF-1α activity is commonly diminished by chromosomal deletion in ccRCCs, and increased HIF-1 activity reduces tumor burden in xenograft tumor models. Conversely, polymorphisms at the HIF-2α gene locus predispose to the development of ccRCCs, and HIF-2α promotes tumor growth. Genetic studies have revealed a prominent role for chromatin-modifying enzyme genes in ccRCC, and these may further modulate specific aspects of the HIF response. This suggests that, rather than global activation of HIF, specific components of the response are important in promoting kidney cancer. Some of these processes are already targets for current therapeutic strategies, and further dissection of this pathway might yield novel methods of treating RCC. In contrast to many tumor types, HIF-1α and HIF-2α have opposing effects in ccRCC biology, with HIF-1α acting as a tumor suppressor and HIF-2α acting as an oncogene. The overall effect of VHL inactivation will depend on fine-tuning of the HIF response. High levels of hypoxia-inducible transcription factors (HIF) are

  2. Methylseleninic acid downregulates hypoxia-inducible factor-1α in invasive prostate cancer.

    Science.gov (United States)

    Sinha, Indu; Null, Kevin; Wolter, William; Suckow, Mark A; King, Tonya; Pinto, John T; Sinha, Raghu

    2012-03-15

    Alternative strategies are needed to control growth of advanced and hormone refractory prostate cancer. In this regard, we investigated the efficacy of methylseleninic acid (MSeA), a penultimate precursor to the highly reactive selenium metabolite, methylselenol, to inhibit growth of invasive and hormone refractory rat (PAIII) and human (PC-3 and PC-3M) prostate cancer cells. Our results demonstrate that MSeA inhibits PAIII cell growth in vitro as well as reduces weights of tumors generated by PAIII cells treated ex vivo. A significant reduction in the number of metastatic lung foci by MSeA treatment was also noted in Lobund-Wistar rats. The PAIII cells along with PC-3, DU145 and PC-3M cells undergo apoptosis after MSeA treatments in both normoxia and hypoxia. Treatment of metastatic rat and human prostate cancer cell lines with MSeA decreased hypoxia-inducible factor-1α (HIF-1α) levels in a dose-dependent manner. Additionally, HIF-1α transcription activity both in normoxic and hypoxic conditions is reduced after MSeA treatment of prostate cancer cells. Furthermore, VEGF and GLUT1, downstream targets of HIF-1α, were also reduced in prostate cancer cells after MSeA treatment. Our study illustrates the efficacy of MSeA in controlling growth of hormone refractory prostate cancer by downregulating HIF-1α, which is possibly occurring through stabilization or increase in prolyl hydroxylase activity. Copyright © 2011 UICC.

  3. Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis

    Directory of Open Access Journals (Sweden)

    Patrick Frost

    2013-01-01

    Full Text Available We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

  4. Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERβ or GPR30 signalling.

    Science.gov (United States)

    Rzemieniec, J; Litwa, E; Wnuk, A; Lason, W; Gołas, A; Krzeptowski, W; Kajta, M

    2015-02-01

    Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERβ) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erβ and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erβ mRNA expression level were in parallel with ERβ immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERβ or GPR30 in

  5. Structural and functional analysis of coral Hypoxia Inducible Factor.

    Directory of Open Access Journals (Sweden)

    Didier Zoccola

    Full Text Available Tissues of symbiotic Cnidarians are exposed to wide, rapid and daily variations of oxygen concentration. Indeed, during daytime, intracellular O2 concentration increases due to symbiont photosynthesis, while during night, respiration of both host cells and symbionts leads to intra-tissue hypoxia. The Hypoxia Inducible Factor 1 (HIF-1 is a heterodimeric transcription factor used for maintenance of oxygen homeostasis and adaptation to hypoxia. Here, we carried out a mechanistic study of the response to variations of O2 concentrations of the coral model Stylophora pistillata. In silico analysis showed that homologs of HIF-1 α (SpiHIF-1α and HIF-1β (SpiHIF-1β exist in coral. A specific SpiHIF-1 DNA binding on mammalian Hypoxia Response Element (HRE sequences was shown in extracts from coral exposed to dark conditions. Then, we cloned the coral HIF-1α and β genes and determined their expression and transcriptional activity. Although HIF-1α has an incomplete Oxygen-dependent Degradation Domain (ODD relative to its human homolog, its protein level is increased under hypoxia when tested in mammalian cells. Moreover, co-transfection of SpiHIF-1α and β in mammalian cells stimulated an artificial promoter containing HRE only in hypoxic conditions. This study shows the strong conservation of molecular mechanisms involved in adaptation to O2 concentration between Cnidarians and Mammals whose ancestors diverged about 1,200-1,500 million years ago.

  6. Epithelial Barrier Regulation by Hypoxia-Inducible Factor.

    Science.gov (United States)

    Glover, Louise E; Colgan, Sean P

    2017-09-01

    Mucosal tissues represent surfaces that are exposed to the outside world and provide a conduit for internal and external communication. Tissues such as the intestine and the lung are lined by layer(s) of epithelial cells that, when organized in three dimensions, provide a critical barrier to the flux of luminal contents. This selective barrier is provided through the regulated expression of junctional proteins and mucins. Tissue oxygen metabolism is central to the maintenance of homeostasis in the mucosa. In some organs (e.g., the colon), low baseline Po2 determines tissue metabolism and results in basal expression of the transcription factor, hypoxia-inducible factor (HIF), which is enhanced after ischemia/inflammation. Recent studies have indicated that HIF contributes fundamentally to the expression of barrier-related genes and in the regulation of barrier-adaptive responses within the mucosa. Here, we briefly review recent literature on the topic of hypoxia and HIF regulation of barrier in mucosal health and during disease.

  7. New metformin derivative HL156A prevents oral cancer progression by inhibiting the insulin-like growth factor/AKT/mammalian target of rapamycin pathways.

    Science.gov (United States)

    Lam, Thuy Giang; Jeong, Yun Soo; Kim, Soo-A; Ahn, Sang-Gun

    2017-12-29

    Metformin is a biguanide widely prescribed as an antidiabetic drug for type 2 diabetes mellitus patients. The purpose of the present study was to observe the effects of the new metformin derivative, HL156A, on human oral cancer cell and to investigate its possible mechanisms. It was observed that HL156A significantly decreased FaDu and YD-10B cell viability and colony formation in a dose-dependent way. HL156A also markedly reduced wound closure and migration of FaDu and YD-10B cells. We observed that HL156A decreased mitochondrial membrane potential and induced reactive oxygen species (ROS) levels and apoptotic cells with caspase-3 and -9 activation. HL156A inhibited the expression and activation of insulin-like growth factor (IGF)-1 and its downstream proteins, AKT, mammalian target of rapamycin (mTOR), and ERK1/2. In addition, HL156A activated AMP-activated protein kinase/nuclear factor kappa B (AMPK-NF-κB) signaling of FaDu and YD-10B cells. A xenograft mouse model further showed that HL156A suppressed AT84 mouse oral tumor growth, accompanied by down-regulated p-IGF-1, p-mTOR, proliferating cell nuclear antigen (PCNA) and promoted p-AMPK and TUNEL expression. These results suggest the potential value of the new metformin derivative HL156A as a candidate for a therapeutic modality for the treatment of oral cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  8. Impairment of object recognition memory by rapamycin inhibition of mTOR in the amygdala or hippocampus around the time of learning or reactivation.

    Science.gov (United States)

    Jobim, Paulo F C; Pedroso, Thiago R; Werenicz, Aline; Christoff, Raissa R; Maurmann, Natasha; Reolon, Gustavo K; Schröder, Nadja; Roesler, Rafael

    2012-03-01

    The role of the basolateral complex of the amygdala (BLA) in recognition memory remains poorly understood. The mammalian target of rapamycin (mTOR) in the BLA and other brain areas has been implicated in synaptic plasticity and memory. We have recently shown that mTOR signaling in both the BLA and the dorsal hippocampus (DH) is required for formation and reconsolidation of inhibitory avoidance, a fear-motivated memory task. Here we examined the effects of infusions of the mTOR inhibitor rapamycin into the BLA before or after either training or reactivation on retention of novel object recognition (NOR) memory in rats, and compared the effects with those obtained using intra-DH infusions. Male Wistar rats received bilateral infusions of vehicle or rapamycin into the BLA or DH before or after NOR training or reactivation. Rapamycin impaired NOR retention tested 24h after training when given either before or immediately after training into the BLA or DH. Rapamycin also impaired retention measured 24h after reactivation when infused before reactivation into the BLA or DH, or immediately after reactivation into the BLA, but not when given 6h after reactivation into either the BLA or DH. The results suggest that mTOR signaling in the BLA and DH is involved in NOR memory formation and stabilization. Copyright © 2011 Elsevier B.V. All rights reserved.

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

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    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.

  10. Myeloid translocation gene-16 co-repressor promotes degradation of hypoxia-inducible factor 1.

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    Full Text Available The myeloid translocation gene 16 (MTG16 co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1 heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1. Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α.

  11. Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J.; Park, Sung-min; Cai, Dongsheng

    2011-01-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance. PMID:21814490

  12. Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma

    Directory of Open Access Journals (Sweden)

    Hongming Teng

    2015-06-01

    Full Text Available Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.

  13. Resistance to hypoxia-induced, BNIP3-mediated cell death contributes to an increase in a CD133-positive cell population in human glioblastomas in vitro.

    Science.gov (United States)

    Kahlert, Ulf Dietrich; Maciaczyk, Donata; Dai, Fangping; Claus, Rainer; Firat, Elke; Doostkam, Soroush; Bogiel, Tomasz; Carro, Maria Stella; Döbrössy, Mate; Herold-Mende, Christel; Niedermann, Gabriele; Prinz, Marco; Nikkhah, Guido; Maciaczyk, Jaroslaw

    2012-12-01

    In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5'-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5'-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.

  14. [High-altitude hypoxia induces disorders of the brain-endocrine-immune network through activation of corticotropin-releasing factor and its type-1 receptors].

    Science.gov (United States)

    Chen, Xue-Qun; Kong, Fan-Ping; Zhao, Yang; Du, Ji-Zeng

    2012-11-01

    High-altitude hypoxia can induce physiological dysfunction and mountain sickness, but the underlying mechanism is not fully understood. Corticotrophin-releasing factor (CRF) and CRF type-i receptors (CRFR1) are members of the CRF family and the essential controllers of the physiological activity of the hypothalamo-pituitary-adrenal (HPA) axis and modulators of endocrine and behavioral activity in response to various stressors. We have previously found that high-altitude hypoxia induces disorders of the brain-endocrine-immune network through activation of CRF and CRFR1 in the brain and periphery that include activation of the HPA axis in a time- and dose-dependent manner, impaired or improved learning and memory, and anxiety-like behavioral change. Meanwhile, hypoxia induces dysfunctions of the hypothalamo-pituitary-endocrine and immune systems, including suppression of growth and development, as well as inhibition of reproductive, metabolic and immune functions. In contrast, the small mammals that live on the Qinghai-Tibet Plateau alpine meadow display low responsiveness to extreme high-altitude-hypoxia challenge, suggesting well-acclimatized genes and a physiological strategy that developed during evolution through interactions between the genes and environment. All the findings provide evidence for understanding the neuroendocrine mechanisms of hypoxia-induced physiological dysfunction. This review extends these findings.

  15. Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

    Science.gov (United States)

    Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L

    2016-10-04

    Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

  16. Rapamycin Inhibits Expression of Elongation of Very-long-chain Fatty Acids 1 and Synthesis of Docosahexaenoic Acid in Bovine Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Zhixin Guo

    2016-11-01

    Full Text Available Mammalian target of rapamycin complex 1 (mTORC1 is a central regulator of cell growth and metabolism and is sufficient to induce specific metabolic processes, including de novo lipid biosynthesis. Elongation of very-long-chain fatty acids 1 (ELOVL1 is a ubiquitously expressed gene and the product of which was thought to be associated with elongation of carbon (C chain in fatty acids. In the present study, we examined the effects of rapamycin, a specific inhibitor of mTORC1, on ELOVL1 expression and docosahexaenoic acid (DHA, C22:6 n-3 synthesis in bovine mammary epithelial cells (BMECs. We found that rapamycin decreased the relative abundance of ELOVL1 mRNA, ELOVL1 expression and the level of DHA in a time-dependent manner. These data indicate that ELOVL1 expression and DHA synthesis are regulated by mTORC1 in BMECs.

  17. Glutamatergic neurotransmission modulates hypoxia-induced hyperventilation but not anapyrexia

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    Paula P.M. de

    2004-01-01

    Full Text Available The interaction between pulmonary ventilation (V E and body temperature (Tb is essential for O2 delivery to match metabolic rate under varying states of metabolic demand. Hypoxia causes hyperventilation and anapyrexia (a regulated drop in Tb, but the neurotransmitters responsible for this interaction are not well known. Since L-glutamate is released centrally in response to peripheral chemoreceptor stimulation and glutamatergic receptors are spread in the central nervous system we tested the hypothesis that central L-glutamate mediates the ventilatory and thermal responses to hypoxia. We measured V E and Tb in 40 adult male Wistar rats (270 to 300 g before and after intracerebroventricular injection of kynurenic acid (KYN, an ionotropic glutamatergic receptor antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG, a metabotropic glutamatergic receptor antagonist or vehicle (saline, followed by a 1-h period of hypoxia (7% inspired O2 or normoxia (humidified room air. Under normoxia, KYN (N = 5 or MCPG (N = 8 treatment did not affect V E or Tb compared to saline (N = 6. KYN and MCPG injection caused a decrease in hypoxia-induced hyperventilation (595 ± 49 for KYN, N = 7 and 525 ± 84 ml kg-1 min-1 for MCPG, N = 6; P < 0.05 but did not affect anapyrexia (35.3 ± 0.2 for KYN and 34.7 ± 0.4ºC for MCPG compared to saline (912 ± 110 ml kg-1 min-1 and 34.8 ± 0.2ºC, N = 8. We conclude that glutamatergic receptors are involved in hypoxic hyperventilation but do not affect anapyrexia, indicating that L-glutamate is not a common mediator of this interaction.

  18. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    OBJECTIVE: To assess the levels and spectrum of mitochondrial DNA (mtDNA) point mutations in synovial tissue from patients with inflammatory arthritis in relation to in vivo hypoxia and oxidative stress levels. METHODS: Random Mutation Capture assay was used to quantitatively evaluate alterations of the synovial mitochondrial genome. In vivo tissue oxygen levels (tPO(2)) were measured at arthroscopy using a Licox probe. Synovial expression of lipid peroxidation (4-hydroxynonenal [4-HNE]) and mitochondrial cytochrome c oxidase subunit II (CytcO II) deficiency were assessed by immunohistochemistry. In vitro levels of mtDNA point mutations, reactive oxygen species (ROS), mitochondrial membrane potential, and markers of oxidative DNA damage (8-oxo-7,8-dihydro-2\\'-deoxyguanine [8-oxodG]) and lipid peroxidation (4-HNE) were determined in human synoviocytes under normoxia and hypoxia (1%) in the presence or absence of superoxide dismutase (SOD) or N-acetylcysteine (NAC) or a hydroxylase inhibitor (dimethyloxalylglycine [DMOG]). Patients were categorized according to their in vivo tPO(2) level (<20 mm Hg or >20 mm Hg), and mtDNA point mutations, immunochemistry features, and stress markers were compared between groups. RESULTS: The median tPO(2) level in synovial tissue indicated significant hypoxia (25.47 mm Hg). Higher frequency of mtDNA mutations was associated with reduced in vivo oxygen tension (P = 0.05) and with higher synovial 4-HNE cytoplasmic expression (P = 0.04). Synovial expression of CytcO II correlated with in vivo tPO(2) levels (P = 0.03), and levels were lower in patients with tPO(2) <20 mm Hg (P < 0.05). In vitro levels of mtDNA mutations, ROS, mitochondrial membrane potential, 8-oxo-dG, and 4-HNE were higher in synoviocytes exposed to 1% hypoxia (P < 0.05); all of these increased levels were rescued by SOD and DMOG and, with the exception of ROS, by NAC. CONCLUSION: These findings demonstrate that hypoxia-induced mitochondrial dysfunction drives

  19. Hypobaric hypoxia induced arginase expression limits nitric oxide availability and signaling in rodent heart.

    Science.gov (United States)

    Singh, Manjulata; Padhy, Gayatri; Vats, Praveen; Bhargava, Kalpana; Sethy, Niroj Kumar

    2014-06-01

    This study was aimed to evaluate regulation of cardiac arginase expression during hypobaric hypoxia and subsequent effect on nitric oxide availability and signaling. Rats were exposed to hypobaric hypoxia (282mmHg for 3h) and ARG1 expression was monitored. The expression levels of eNOS and eNOS(Ser1177) were determined by Western blotting, cGMP levels were measured by ELISA and amino acid concentrations were measured by HPLC analysis. Transcription regulation of arginase was monitored by chromatin immunoprecipitation (ChIP) assay with anti-c-Jun antibody for AP-1 consensus binding site on ARG1 promoter. Arginase activity was inhibited by intra-venous dose of N-(ω)-hydroxy-nor-l-arginine (nor-NOHA) prior to hypoxia exposure and subsequent effect on NO availability and oxidative stress were evaluated. Hypobaric hypoxia induced cardiac arginase expression by recruiting c-Jun to AP-1 binding site on ARG1 promoter. This increased expression redirected l-arginine towards arginase and resulted in limited endothelial nitric oxide synthase (eNOS) activity, nitric oxide (NO) availability and cGMP mediated signaling. Inhibition of arginase restored the eNOS activity, promoted cardiac NO availability and ameliorated peroxynitrite formation during hypoxia. Hypoxic induced arginase under transcription control of AP-1 reciprocally regulates eNOS activity and NO availability in the heart. This also results in cardiac oxidative stress. This study provides understanding of hypoxia-mediated transcriptional regulation of arginase expression in the heart and its subsequent effect on eNOS activity, NO availability and signaling as well as cardiac oxidative stress. This information will support the use of arginase inhibitors as therapeutics for pathological hypoxia. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. 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.

  1. Absence of COX-2 exacerbates hypoxia-induced pulmonary hypertension and enhances contractility of vascular smooth muscle cells

    Science.gov (United States)

    Fredenburgh, Laura E.; Liang, Olin D.; Macias, Alvaro A.; Polte, Thomas R.; Liu, Xiaoli; Riascos, Dario F.; Chung, Su Wol; Schissel, Scott L.; Ingber, Donald E.; Mitsialis, S. Alex; Kourembanas, Stella; Perrella, Mark A.

    2008-01-01

    Background Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMC) during hypoxia and may play a protective role in the lung’s response to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia. Methods and Results To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2 deficient mice to a model of chronic normobaric hypoxia. COX-2 null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling following hypoxia. Pulmonary vascular remodeling in COX-2 deficient mice was characterized by PASMC hypertrophy, but not increased proliferation. Furthermore, COX-2 deficient mice had significant upregulation of the ET-1 receptor (ETAR) in the lung following hypoxia. Similarly, selective pharmacologic inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ETAR expression in pulmonary arterioles. Absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2 deficient PASMC with iloprost, a prostaglandin (PG) I2 analog, as well as PGE2, abrogated the potent contractile response to hypoxia and restored the wild-type phenotype. Conclusions Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling is exacerbated in the absence of COX-2 with enhanced ETA receptor expression and increased PASMC hypertrophy. COX-2 deficient PASMC have a maladaptive response to hypoxia manifested by exaggerated contractility which may be rescued by either COX-2-derived PGI2 or PGE2. PMID:18391113

  2. NOX2 Antisense Attenuates Hypoxia-Induced Oxidative Stress and Apoptosis in Cardiomyocyte.

    Science.gov (United States)

    Yu, Bo; Meng, Fanbo; Yang, Yushuang; Liu, Dongna; Shi, Kaiyao

    2016-01-01

    Heart ischemia is a hypoxia related disease. NOX2 and HIF-1α proteins were increased in cardiomyocytes after acute myocardial infarction. However, the relationship of the hypoxia-induced HIF-1α. NOX2-derived oxidative stress and apoptosis in cardiomyocyte remains unclear. In the current study, we use NOX2 antisense strategy to investigate the role of NOX2 in hypoxia-induced oxidative stress and apoptosis in rat cardiomyocytes. Here, we show that transduction of ADV-NOX2-AS induces potent silencing of NOX2 in cardiomyocytes, and resulting in attenuation of hypoxia-induced oxidative stress and apoptosis. This study indicates the potential of antisense-based therapies and validates NOX2 as a potent therapeutic candidate for heart ischemia.

  3. Hypoxia induces oncogene yes-associated protein 1 nuclear translocation to promote pancreatic ductal adenocarcinoma invasion via epithelial-mesenchymal transition.

    Science.gov (United States)

    Wei, Honglong; Xu, Zongzhen; Liu, Feng; Wang, Fuhai; Wang, Xin; Sun, Xueying; Li, Jie

    2017-05-01

    Pancreatic ductal adenocarcinoma is one of the most lethal cancers. The Hippo pathway is involved in tumorigenesis and remodeling of tumor microenvironments. Hypoxia exists in the microenvironment of solid tumors, including pancreatic ductal adenocarcinoma and plays a vital role in tumor progression and metastasis. However, it remains unclear how hypoxia interacts with the Hippo pathway to regulate these events. In this study, expressions of yes-associated protein 1 and hypoxia-inducible factor-1α were found to be elevated in pancreatic ductal adenocarcinoma samples compared with those in matched adjacent non-tumor samples. Moreover, hypoxia-inducible factor-1α expression was positively correlated with yes-associated protein 1 level in pancreatic ductal adenocarcinoma tissues. The higher expression of nuclear yes-associated protein 1 was associated with poor histological grade and prognosis for pancreatic ductal adenocarcinoma patients. In vitro, yes-associated protein 1 was highly expressed in pancreatic ductal adenocarcinoma cells. Depletion of yes-associated protein 1 inhibited the invasion of pancreatic ductal adenocarcinoma cells via downregulation of Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-13, and upregulation of E-cadherin. In addition, hypoxia promoted the invasion of pancreatic ductal adenocarcinoma cells via regulating the targeted genes. Hypoxia also deactivated the Hippo pathway and induced yes-associated protein 1 nuclear translocation. Furthermore, depletion of yes-associated protein 1 or hypoxia-inducible factor-1α suppressed the invasion of pancreatic ductal adenocarcinoma cells under hypoxia. Mechanism studies showed that nuclear yes-associated protein 1 interacted with hypoxia-inducible factor-1α and activated Snail transcription to participate in epithelial-mesenchymal transition-mediated and matrix metalloproteinase-mediated remodeling of tumor microenvironments. Collectively, yes-associated protein 1 is an

  4. Modulation of Hypoxia-Induced Pulmonary Vascular Leakage in Rats by Seabuckthorn (Hippophae rhamnoides L.

    Directory of Open Access Journals (Sweden)

    Jayamurthy Purushothaman

    2011-01-01

    Full Text Available Cerebral and pulmonary syndromes may develop in unacclimatized individuals shortly after ascent to high altitude resulting in high altitude illness, which may occur due to extravasation of fluid from intra to extravascular space in the brain, lungs and peripheral tissues. The objective of the present study was to evaluate the potential of seabuckthorn (SBT (Hippophae rhamnoides L. leaf extract (LE in curtailing hypoxia-induced transvascular permeability in the lungs by measuring lung water content, leakage of fluorescein dye into the lungs and further confirmation by quantitation of albumin and protein in the bronchoalveolar lavage fluid (BALF. Exposure of rats to hypoxia caused a significant increase in the transvascular leakage in the lungs. The SBT LE treated animals showed a significant decrease in hypoxia-induced vascular permeability evidenced by decreased water content and fluorescein leakage in the lungs and decreased albumin and protein content in the BALF. The SBT extract was also able to significantly attenuate hypoxia-induced increase in the levels of proinflammatory cytokines and decrease hypoxia-induced oxidative stress by stabilizing the levels of reduced glutathione and antioxidant enzymes. Pretreatment of the extract also resulted in a significant decrease in the circulatory catecholamines and significant increase in the vasorelaxation of the pulmonary arterial rings as compared with the controls. Further, the extract significantly attenuated hypoxia-induced increase in the VEGF levels in the plasma, BALF (ELISA and lungs (immunohistochemistry. These observations suggest that SBT LE is able to provide significant protection against hypoxia-induced pulmonary vascular leakage.

  5. 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

  6. Red blood cell storage increases hypoxia-induced nitric oxide bioavailability and methemoglobin formation in vitro and in vivo

    NARCIS (Netherlands)

    Almac, Emre; Bezemer, Rick; Hilarius-Stokman, Petra M.; Goedhart, Peter; de Korte, Dirk; Verhoeven, Arthur J.; Ince, Can

    2014-01-01

    In this study we investigated whether storage of red blood cells (RBCs) leads to alterations in nitrite reductase activity, hence in altered hypoxia-induced nitric oxide (NO) bioavailability and methemoglobin formation. Hypoxia-induced NO bioavailability and methemoglobin formation were measured in

  7. Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets

    Science.gov (United States)

    Cheng, Kim; Ho, Kenneth; Stokes, Rebecca; Scott, Christopher; Lau, Sue Mei; Hawthorne, Wayne J.; O’Connell, Philip J.; Loudovaris, Thomas; Kay, Thomas W.; Kulkarni, Rohit N.; Okada, Terumasa; Wang, Xiaohui L.; Yim, Sun Hee; Shah, Yatrik; Grey, Shane T.; Biankin, Andrew V.; Kench, James G.; Laybutt, D. Ross; Gonzalez, Frank J.; Kahn, C. Ronald; Gunton, Jenny E.

    2010-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1α protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1α is required for normal β cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1α protein is present at low levels in mouse and human normoxic β cells and islets. Decreased levels of HIF-1α impaired glucose-stimulated ATP generation and β cell function. C57BL/6 mice with β cell–specific Hif1a disruption (referred to herein as β-Hif1a-null mice) exhibited glucose intolerance, β cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1α levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in β-Hif1a-null mice. Increasing HIF-1α levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1α was bound to the Arnt promoter in a mouse β cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1α in β cell reserve and regulation of ARNT expression and demonstrate that HIF-1α is a potential therapeutic target for the β cell dysfunction of T2D. PMID:20440072

  8. Notch signaling modulates hypoxia-induced neuroendocrine differentiation of human prostate cancer cells.

    Science.gov (United States)

    Danza, Giovanna; Di Serio, Claudia; Rosati, Fabiana; Lonetto, Giuseppe; Sturli, Niccolò; Kacer, Doreen; Pennella, Antonio; Ventimiglia, Giuseppina; Barucci, Riccardo; Piscazzi, Annamaria; Prudovsky, Igor; Landriscina, Matteo; Marchionni, Niccolò; Tarantini, Francesca

    2012-02-01

    Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription. ©2011 AACR.

  9. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

    Directory of Open Access Journals (Sweden)

    Apolinario Rosa M

    2009-08-01

    Full Text Available Abstract Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP, vault poly(ADP-ribose polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022. Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003. Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

  10. Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Satwiko, Muhammad Gahan [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Ikeda, Koji [Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan); Nakayama, Kazuhiko [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Yagi, Keiko [Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan); Hocher, Berthold [Institute for Nutritional Science, University of Potsdam, Potsdam (Germany); Hirata, Ken-ichi [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Emoto, Noriaki, E-mail: emoto@med.kobe-u.ac.jp [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan)

    2015-09-25

    Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH. - Highlights: • Role of endothelin-1 in pulmonary arterial hypertension (PAH) was investigated. • The endothelin-1 transgenic (ETTG) and wild type (WT) mice were analyzed. • ETTG mice spontaneously developed PAH under normoxia conditions. • SU5416 further aggravated PAH in ETTG mice. • Enhanced endothelin system activity could be a causative factor in

  11. NOTCH SIGNALLING MODULATES HYPOXIA-INDUCED NEUROENDOCRINE DIFFERENTIATION OF HUMAN PROSTATE CANCER CELLS

    Science.gov (United States)

    Danza, Giovanna; Di Serio, Claudia; Rosati, Fabiana; Lonetto, Giuseppe; Sturli, Niccolò; Kacer, Doreen; Pennella, Antonio; Ventimiglia, Giuseppina; Barucci, Riccardo; Piscazzi, Annamaria; Prudovsky, Igor; Landriscina, Matteo; Marchionni, Niccolò; Tarantini, Francesca

    2012-01-01

    Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation has been associated with tumor progression, poor prognosis and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavourable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells, in vitro. Results exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent down regulation of Notch-mediated signalling, as demonstrated by reduced levels of the Notch target genes, Hes1 and Hey1. Neuroendocrine differentiation was promoted by attenuation of Hes1 transcription, as cells expressing a dominant negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia down regulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen independent cell lines, PC3 and Du145, it did not change the extent of NE differentiation in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Conclusions hypoxia induces neuroendocrine differentiation of LNCaP cells in vitro, which appears to be driven by the inhibition of Notch signalling with subsequent down-regulation of Hes1 transcription. PMID:22172337

  12. Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

    Directory of Open Access Journals (Sweden)

    Min Yu

    Full Text Available Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

  13. Methanolic extract of onion (Allium cepa) attenuates ischemia/hypoxia-induced apoptosis in cardiomyocytes via antioxidant effect.

    Science.gov (United States)

    Park, Sok; Kim, Mi-Young; Lee, Dong Ha; Lee, Soo Hwan; Baik, Eun Joo; Moon, Chang-Hyun; Park, Se Won; Ko, Eun Young; Oh, Sei-Ryang; Jung, Yi-Sook

    2009-06-01

    Although there is growing awareness of the beneficial potential of onion intake to lower the risk of cardiovascular disease, there is little information about the effect of onion on ischemic heart injury, one of the most common cardiovascular diseases. This study investigates the effect of the methanol-soluble extract of onion on ischemic injury in heart-derived H9c2 cells in vitro and in rat hearts in vivo. The underlying mechanism is also investigated. To evaluate the effect of onion on ischemia-induced cell death, LDH release and TUNEL-positivity were assessed in H9c2 cells, and the infarct size was measured in a myocardial infarct model. To investigate the mechanism of the cardioprotection by onion, the reactive oxygen species (ROS) level and the mitochondrial membrane potential (DeltaPsi(m)) were measured using an imaging technique; the caspase-3 activity was assayed, and Western blotting was performed to examine cytochrome c release in H9c2 cells. The methanolic extract of onion had a preventive effect on ischemia/hypoxia-induced apoptotic death in H9c2 cells in vitro and in rat heart in vivo. The onion extract (0.05 g/ml) inhibited the elevation of the ROS, mitochondrial membrane depolarization, cytochrome c release and caspase-3 activation during hypoxia in H9c2 cells. In the in vivo rat myocardial infarction model, onion extract (10 g/kg) significantly reduced the infarct size, the apoptotic cell death of the heart and the plasma MDA level. In conclusion, the results of this study suggest that the methanolic extract of onion attenuates ischemia/hypoxia-induced apoptosis in heart-derived H9c2 cells in vitro and in rat hearts in vivo, through, at least in part, an antioxidant effect.

  14. 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

  15. The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

    Science.gov (United States)

    Mukaida, Shiho; Ogawa, Takumi; Ohishi, Kazuko; Tanizawa, Yasuhiro; Ohta, Daisaku; Arita, Masanori

    2016-06-01

    Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.

  16. 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.

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

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    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.

  18. Hypoxia-inducible factors - regulation, role and comparative aspects in tumourigenesis

    DEFF Research Database (Denmark)

    Hansen, A E; Kristensen, A T; Law, I

    2011-01-01

    Hypoxia-inducible factors (HIFs) play a key role in the cellular response experienced in hypoxic tumours, mediating adaptive responses that allow hypoxic cells to survive in the hostile environment. Identification and understanding of tumour hypoxia and the influence on cellular processes carries...

  19. Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery

    NARCIS (Netherlands)

    Christensen, Lane V.; Christensen, L.; Chang, Chien-Wen; Yockman, James W.; Conners, Rafe; Jackson, Heidi; Zhong, Zhiyuan; Feijen, Jan; Bull, David A.; Kim, Sung Wan

    2007-01-01

    Delivery of the hypoxia-inducible vascular endothelial growth factor (RTP-VEGF) plasmid using a novel reducible disulfide poly(amido ethylenediamine) (SS-PAED) polymer carrier was studied in vitro and in vivo. In vitro transfection of primary rat cardiomyoblasts (H9C2) showed SS-PAED at a weighted

  20. Association of thymidylate synthase and hypoxia inducible factor-1alpha DNA polymorphisms with pancreatic cancer.

    Science.gov (United States)

    Ruiz-Tovar, Jaime; Fernandez-Contreras, Maria Encarnación; Martín-Perez, Elena; Gamallo, Carlos

    2012-01-01

    Thymidylate synthase and hypoxia inducible factor-1α play a central role in the control of tumor progression. In the present study, we investigated the effect of three DNA polymorphisms within the thymidylate synthase gene and two within hypoxia inducible factor-1α on the prognosis of pancreatic cancer. A retrospective study was performed in 59 patients diagnosed with invasive ductal adenocarcinoma of the pancreas and 159 healthy volunteers. The studied DNA polymorphisms were a variable tandem repeat of 28 bp (rs45445694), a G/C single nucleotide polymorphism (rs34743033), and a deletion of 6 bp (ins1494del 6bp; rs34489327) within the thymidylate synthase gene and C1772T and G1790A single nucleotide polymorphisms within hypoxia inducible factor-1α (rs11549465 and rs11549467, respectively) . Variable tandem repeats were determined by specific polymerase chain reaction, whereas thymidylate synthase single nucleotide polymorphism G/C, ins1494del 6pb, and hypoxia inducible factor-1α polymorphisms were identified by polymerase chain reaction and RFLP. Thymidylate synthase and hypoxia inducible factor-1α genotype distributions in patients and healthy volunteers were determined. The impact of the polymorphisms on clinico-pathological variables, including survival, was also studied. The frequency of carriers of the variant del6bp allele was significantly higher among patients (70.0% vs 51.0% of healthy donors, P = 0.02); 42% of male patients were homozygous 2R/2R vs 13.6% of females (P = 0.03), but differences regarding gender were not observed among healthy volunteers. Concerning hypoxia inducible factor-1α C1772T and G1790A single nucleotide polymorphisms, the rates of variant T/T and A/A homozygous genotypes were significantly elevated among patients (18.6% vs 5.3%, P = 0.001, and 5.1% vs none, P = 0.021 respectively). In our study, the variant del14946bp allele within the thymidylate synthase gene, and TT and AA genotypes of C1772T and G1790A hypoxia inducible

  1. Rapamycin is neuroprotective in a rat chronic hypertensive glaucoma model.

    Directory of Open Access Journals (Sweden)

    Wenru Su

    Full Text Available Glaucoma is a leading cause of irreversible blindness. Injury of retinal ganglion cells (RGCs accounts for visual impairment of glaucoma. Here, we report rapamycin protects RGCs from death in experimental glaucoma model and the underlying mechanisms. Our results showed that treatment with rapamycin dramatically promote RGCs survival in a rat chronic ocular hypertension model. This protective action appears to be attributable to inhibition of neurotoxic mediators release and/or direct suppression of RGC apoptosis. In support of this mechanism, in vitro, rapamycin significantly inhibits the production of NO, TNF-α in BV2 microglials by modulating NF-κB signaling. In experimental animals, treatment with rapamycin also dramatically inhibited the activation of microglials. In primary RGCs, rapamycin was capable of direct suppression the apoptosis of primary RGCs induced by glutamate. Mechanistically, rapamycin-mediated suppression of RGCs apoptosis is by sparing phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity in cell and animal model. These results demonstrate that rapamycin is neuroprotective in experimental glaucoma, possibly via decreasing neurotoxic releasing and suppressing directly apoptosis of RGCs.

  2. The mTOR inhibitor rapamycin has limited acute anticonvulsant effects in mice.

    Directory of Open Access Journals (Sweden)

    Adam L Hartman

    Full Text Available The mammalian target of rapamycin (mTOR pathway integrates signals from different nutrient sources, including amino acids and glucose. Compounds that inhibit mTOR kinase activity such as rapamycin and everolimus can suppress seizures in some chronic animal models and in patients with tuberous sclerosis. However, it is not known whether mTOR inhibitors exert acute anticonvulsant effects in addition to their longer term antiepileptogenic effects. To gain insights into how rapamycin suppresses seizures, we investigated the anticonvulsant activity of rapamycin using acute seizure tests in mice.Following intraperitoneal injection of rapamycin, normal four-week-old male NIH Swiss mice were evaluated for susceptibility to a battery of acute seizure tests similar to those currently used to screen potential therapeutics by the US NIH Anticonvulsant Screening Program. To assess the short term effects of rapamycin, mice were seizure tested in ≤ 6 hours of a single dose of rapamycin, and for longer term effects of rapamycin, mice were tested after 3 or more daily doses of rapamycin.The only seizure test where short-term rapamycin treatment protected mice was against tonic hindlimb extension in the MES threshold test, though this protection waned with longer rapamycin treatment. Longer term rapamycin treatment protected against kainic acid-induced seizure activity, but only at late times after seizure onset. Rapamycin was not protective in the 6 Hz or PTZ seizure tests after short or longer rapamycin treatment times. In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice.The efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.

  3. Overexpression of Hypoxia-Inducible Factor-1α Exacerbates Endothelial Barrier Dysfunction Induced by Hypoxia

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    Pei Wang

    2013-09-01

    Full Text Available Background/Aims: The mechanisms involved in endothelial barrier dysfunction induced by hypoxia are incompletely understood. There is debate about the role of hypoxia-inducible factor-1α (HIF-1α in endothelial barrier disruption. The aim of this study was to investigate the effect of genetic overexpression of HIF-1α on barrier function and the underlying mechanisms in hypoxic endothelial cells. Methods: The plasmid pcDNA3.1/V5-His-HIF-1α was stably transfected into human endothelial cells. The cells were exposed to normoxia or hypoxia. The mRNA and protein expressions of HIF-1α were detected by RT-PCR and Western blot respectively. The barrier function was assessed by measuring the transendothelial electrical resistance (TER. The Western blot analysis was used to determine the protein expression of glucose transporter-1 (GLUT-1, zonular occludens-1 (ZO-1, occludin, and myosin light chain kinase (MLCK in endothelial cells. The mRNA expression of proinflammatory cytokines was detected by qRT-PCR. Results: Genetic overexpression of HIF-1α significantly increased the mRNA and protein expression of HIF-1α in endothelial cells. The overexpression of HIF-1α enhanced the hypoxia-induced increase of HIF-1α and GLUT-1 protein expression. HIF-1α overexpression not only exacerbated hypoxia-induced endothelial barrier dysfunction but also augmented hypoxia-induced up-regulation of MLCK protein expression. HIF-1α overexpression also enhanced IL-1β, IL-6 and TNF-α mRNA expression. Conclusion: We provide evidence that genetic overexpression of HIF-1α aggravates the hypoxia-induced endothelial barrier dysfunction via enhancing the up-regulation of MLCK protein expression caused by hypoxia, suggesting a potential role for HIF-1α in the pathogenesis of endothelial barrier dysfunction in hypoxia.

  4. THE EFFECT OF RAPAMYCIN ON SECRETORY ACTIVITY OF THE RABBIT OVARIAN FRAGMENTS

    Directory of Open Access Journals (Sweden)

    Sushmita Nath

    2012-02-01

    Full Text Available The aim of our study was to examine the effect of rapamycin on secretory activity of the rabbit ovarian fragments. The secretion of steroid (progesterone, testosterone, estradiol and peptide (prolactine hormones by ovarian fragments after rapamycin addition at the doses 0, 1, 10, 100 μg.ml-1 was determined. Fragments were incubated with rapamycin for 48 hours. Hormones were determinated by RIA. The experimental data showed that, addition of rapamycin did not affect progesterone and prolactine release (at all doses. Estradiol secretion was inhibited by rapamycin at the doses of 1, 10 and 100 μg.ml-1. Testosterone was inhibited by the rapamycin at the doses of 1 and 10 μg.ml-1 but not at 100 μg.ml-1. In conclusion, our results suggest a direct effect of rapamycin on ovarian functionsand a possible involvement in the regulation of steroidogenesis.

  5. Rapamycin carbonate esters

    OpenAIRE

    Rhodes, A; Sandhu, S S; Onis, J. E; McKendrick, John

    2009-01-01

    Certain embodiments include carbonate esters of rapamycin at position 42 that are synthesized by a lipase catalyzed regio-specific process. These compounds or a pharmaceutically acceptable salt thereof are useful in the treatment of organ and tissue transplant rejection, autoimmune disease, proliferative disorder, restenosis, cancer, or microbial infection.

  6. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.

    Science.gov (United States)

    Blanco, F F; Jimbo, M; Wulfkuhle, J; Gallagher, I; Deng, J; Enyenihi, L; Meisner-Kober, N; Londin, E; Rigoutsos, I; Sawicki, J A; Risbud, M V; Witkiewicz, A K; McCue, P A; Jiang, W; Rui, H; Yeo, C J; Petricoin, E; Winter, J M; Brody, J R

    2016-05-01

    Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in

  7. Anti-angiogenic and anti-tumor activity of Bavachinin by targeting hypoxia-inducible factor-1α.

    Science.gov (United States)

    Nepal, Manoj; Choi, Hwa Jung; Choi, Bo-Yun; Kim, Se Lim; Ryu, Jae-Ha; Kim, Do Hee; Lee, Young-Hoon; Soh, Yunjo

    2012-09-15

    Hypoxia-inducible factor-1 (HIF-1) consists of two subunits, the HIF-1β, which is constitutively expressed, and HIF-1α, which is oxygen-responsive. HIF-1α is over-expressed in response to hypoxia, increasing transcriptional activity linked to tumor progression, angiogenesis, metastasis, and invasion. This study aimed to demonstrate that the natural compound, Bavachinin, has potent anti-angiogenic activity in vitro and in vivo. Bavachinin inhibited increases in HIF-1α activity in human KB carcinoma (HeLa cell derivative) and human HOS osteosarcoma cells under hypoxia in a concentration-dependent manner, probably by enhancing the interaction between von Hippel-Lindau (VHL) and HIF-1α. Furthermore, Bavachinin decreased transcription of genes associated with angiogenesis and energy metabolism that are regulated by HIF-1, such as vascular endothelial growth factors (VEGF), Glut 1 and Hexokinase 2. Bavachinin also inhibited tube formation in human umbilical vein endothelial cells (HUVECs) as well as in vitro migration of KB cells. In vivo studies showed that injecting Bavachinin thrice weekly for four weeks significantly reduced tumor volume and CD31 expression in nude mice with KB xenografts. These data indicate that Bavachinin could be used as a therapeutic agent for inhibiting tumor angiogenesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Involvement of Ca2+-activated K+channel 3.1 in hypoxia-induced pulmonary arterial hypertension and therapeutic effects of TRAM-34 in rats.

    Science.gov (United States)

    Guo, Shujin; Shen, Yongchun; He, Guangming; Wang, Tao; Xu, Dan; Wen, Fuqiang

    2017-08-31

    Pulmonary artery hypertension (PAH) is an incurable disease associated with the proliferation of pulmonary artery smooth muscle cells (PASMCs) and vascular remodeling. The present study examined whether TRAM-34, a highly selective blocker of calcium-activated potassium channel 3.1 (Kca3.1), can help prevent such hypertension by reducing proliferation in PASMCs. Rats were exposed to hypoxia (10% O 2 ) for 3 weeks and treated daily with TRAM-34 intraperitoneally from the first day of hypoxia. Animals were killed and examined for vascular hypertrophy, Kca3.1 expression, and downstream signaling pathways. In addition, primary cultures of rat PASMCs were exposed to hypoxia (3% O 2 ) or normoxia (21% O 2 ) for 24 h in the presence of TRAM-34 or siRNA against Kca3.1. Activation of cell signaling pathways was examined using Western blot analysis. In animal experiments, hypoxia triggered significant medial hypertrophy of pulmonary arterioles and right ventricular hypertrophy, and it significantly increased pulmonary artery pressure, Kca3.1 mRNA levels and ERK/p38 MAP kinase signaling. These effects were attenuated in the presence of TRAM-34. In cell culture experiments, blocking Kca3.1 using TRAM-34 or siRNA inhibited hypoxia-induced ERK/p38 signaling. Kca3.1 may play a role in the development of PAH by activating ERK/p38 MAP kinase signaling, which may then contribute to hypoxia-induced pulmonary vascular remodeling. TRAM-34 may protect against hypoxia-induced PAH. © 2017 The Author(s).

  9. Role of nitric oxide in hypoxia-induced hyperventilation and hypothermia: participation of the locus coeruleus

    Directory of Open Access Journals (Sweden)

    Fabris G.

    1999-01-01

    Full Text Available Hypoxia elicits hyperventilation and hypothermia, but the mechanisms involved are not well understood. The nitric oxide (NO pathway is involved in hypoxia-induced hypothermia and hyperventilation, and works as a neuromodulator in the central nervous system, including the locus coeruleus (LC, which is a noradrenergic nucleus in the pons. The LC plays a role in a number of stress-induced responses, but its participation in the control of breathing and thermoregulation is unclear. Thus, in the present study, we tested the hypothesis that LC plays a role in the hypoxia-induced hypothermia and hyperventilation, and that NO is involved in these responses. Electrolytic lesions were performed bilaterally within the LC in awake unrestrained adult male Wistar rats weighing 250-350 g. Body temperature and pulmonary ventilation (VE were measured. The rats were divided into 3 groups: control (N = 16, sham operated (N = 7 and LC lesioned (N = 19, and each group received a saline or an NG-nitro-L-arginine methyl ester (L-NAME, 250 µg/µl intracerebroventricular (icv injection. No significant difference was observed between control and sham-operated rats. Hypoxia (7% inspired O2 caused hyperventilation and hypothermia in both control (from 541.62 ± 35.02 to 1816.18 ± 170.7 and 36.3 ± 0.12 to 34.4 ± 0.09, respectively and LC-lesioned rats (LCLR (from 694.65 ± 63.17 to 2670.29 ± 471.33 and 36 ± 0.12 to 35.3 ± 0.12, respectively, but the increase in VE was higher (P<0.05 and hypothermia was reduced (P<0.05 in LCLR. L-NAME caused no significant change in VE or in body temperature under normoxia, but abolished both the hypoxia-induced hyperventilation and hypothermia. Hypoxia-induced hyperventilation was reduced in LCLR treated with L-NAME. L-NAME also abolished the hypoxia-induced hypothermia in LCLR. The present data indicate that hypoxia-induced hyperventilation and hypothermia may be related to the LC, and that NO is involved in these responses.

  10. Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat (FG-4592) for the Treatment of Anemia in Patients with CKD

    National Research Council Canada - National Science Library

    Provenzano, Robert; Besarab, Anatole; Sun, Chao H; Diamond, Susan A; Durham, John H; Cangiano, Jose L; Aiello, Joseph R; Novak, James E; Lee, Tyson; Leong, Robert; Roberts, Brian K; Saikali, Khalil G; Hemmerich, Stefan; Szczech, Lynda A; Yu, Kin-Hung Peony; Neff, Thomas B

    2016-01-01

    Roxadustat (FG-4592), an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis, regulates iron metabolism, and reduces hepcidin, was evaluated in this phase 2b study for safety...

  11. Further insights into the mechanism of hypoxia-induced NFκB. [corrected].

    Science.gov (United States)

    Melvin, Andrew; Mudie, Sharon; Rocha, Sonia

    2011-03-15

    The cellular response to hypoxia relies on the activation of a specific transcriptional program. Although, most of the attention is focused on the transcription factor HIF, other transcription factors are also activated in hypoxia. We have recently described the mechanism for hypoxia induced NFκB. We have demonstrated the crucial dependency on the IKK complex as well as in the upstream IKK kinase TAK1. TAK1 and IKK activation is dependent upon the calcium calmodulin kinase, CaMK2 and requires Ubc13 as the E2 ubiquitin conjugation enzyme. We report a role for XIAP as the possible E3-ubiquitin ligase for this system. Interestingly, hypoxia induced IKK mediated phosphorylation of IκBα, does not lead to degradation. Hypoxia prevents IκBα de-sumoylation of Sumo-2/3 chains on critical lysine residues, normally required for K-48 linked polyubiquitination. Our results define a novel pathway regulating NFκB activation.

  12. The Hypoxia-Inducible Factor Pathway, Prolyl Hydroxylase Domain Protein Inhibitors, and Their Roles in Bone Repair and Regeneration

    Directory of Open Access Journals (Sweden)

    Lihong Fan

    2014-01-01

    Full Text Available Hypoxia-inducible factors (HIFs are oxygen-dependent transcriptional activators that play crucial roles in angiogenesis, erythropoiesis, energy metabolism, and cell fate decisions. The group of enzymes that can catalyse the hydroxylation reaction of HIF-1 is prolyl hydroxylase domain proteins (PHDs. PHD inhibitors (PHIs activate the HIF pathway by preventing degradation of HIF-α via inhibiting PHDs. Osteogenesis and angiogenesis are tightly coupled during bone repair and regeneration. Numerous studies suggest that HIFs and their target gene, vascular endothelial growth factor (VEGF, are critical regulators of angiogenic-osteogenic coupling. In this brief perspective, we review current studies about the HIF pathway and its role in bone repair and regeneration, as well as the cellular and molecular mechanisms involved. Additionally, we briefly discuss the therapeutic manipulation of HIFs and VEGF in bone repair and bone tumours. This review will expand our knowledge of biology of HIFs, PHDs, PHD inhibitors, and bone regeneration, and it may also aid the design of novel therapies for accelerating bone repair and regeneration or inhibiting bone tumours.

  13. Isolation of hypoxia-inducible factor 1 (HIF-1) inhibitors from frankincense using a molecularly imprinted polymer.

    Science.gov (United States)

    Lakka, Achillia; Mylonis, Ilias; Bonanou, Sophia; Simos, George; Tsakalof, Andreas

    2011-10-01

    Hypoxia-Inducible Factor 1 (HIF-1), a transcriptional activator, is highly involved in the pathology of cancer. Inhibition of HIF-1 retards tumor growth and enhances treatment efficiency when used in combination with chemo- or radiation therapy. The recent validation of HIF-1 as an important drug target in cancer treatment has stimulated efforts to identify and isolate natural or synthetic HIF-1 inhibitors. In the present study, quercetin, a known inhibitor of HIF-1, was imprinted in a polymer matrix in order to prepare a Molecularly Imprinted Polymer (MIP), which was subsequently used for the selective isolation of new inhibitors from frankincense, a gum resin used as anticancer remedy in traditional medicine. The frankincense components isolated by Solid Phase Extraction on MIP (MIP-SPE), efficiently inhibited the transcriptional activity of HIF-1 and decreased the protein levels of HIF-1α, the regulated subunit of HIF-1. The selective retention of acetyl 11-ketoboswellic acid (AKBA, one of the main bioactive components of frankincense) by MIP led to the revealing of its inhibitory activity on the HIF-1 signaling pathway. AKBA was selectively retained by SPE on the quercetin imprinted polymer, with an imprinting effect of 8.1 ± 4.6. Overall, this study demonstrates the potential of MIP application in the screening, recognition and isolation of new bioactive compounds that aim selected molecular targets, a potential that has been poorly appreciated until.

  14. Hypoxia inducible BHLHB2 is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Weibin; Reiser-Erkan, Carolin; Michalski, Christoph W.; Raggi, Matthias C. [Department of Surgery, Technische Universitaet Muenchen, Munich (Germany); Quan, Liao; Yupei, Zhao [Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Peking (China); Friess, Helmut [Department of Surgery, Technische Universitaet Muenchen, Munich (Germany); Erkan, Mert, E-mail: erkan@chir.med.tu-muenchen.de [Department of Surgery, Technische Universitaet Muenchen, Munich (Germany); Kleeff, Joerg [Department of Surgery, Technische Universitaet Muenchen, Munich (Germany)

    2010-10-22

    cells. Patients with weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p = 0.03). In a multivariable analysis, BHLHB2 staining was an independent prognostic factor (Hazard-Ratio = 2.348, 95% CI = 1.250-4.411, p = 0.008). Conclusions: Hypoxia-inducible BHLHB2 expression is a novel independent prognostic marker in pancreatic cancer patients and indicates increased chemosensitivity towards gemcitabine.

  15. Ancient atmospheres and the evolution of oxygen sensing via the hypoxia-inducible factor in metazoans.

    Science.gov (United States)

    Taylor, Cormac T; McElwain, Jennifer C

    2010-10-01

    Metazoan diversification occurred during a time when atmospheric oxygen levels fluctuated between 15 and 30%. The hypoxia-inducible factor (HIF) is a primary regulator of the adaptive transcriptional response to hypoxia. Although the HIF pathway is highly conserved, its complexity increased during periods when atmospheric oxygen concentrations were increasing. Thus atmospheric oxygen levels may have provided a selection force on the development of cellular oxygen-sensing pathways.

  16. Ascorbic acid does not enhance hypoxia-induced vasodilation in healthy older men.

    Science.gov (United States)

    Pollock, Jonathan P; Patel, Hardikkumar M; Randolph, Brittney J; Heffernan, Matthew J; Leuenberger, Urs A; Muller, Matthew D

    2014-07-01

    In response to hypoxia, a net vasodilation occurs in the limb vasculature in young healthy humans and this is referred to as "hypoxia-induced vasodilation". We performed two separate experiments to determine (1) if hypoxia-induced forearm vasodilation is impaired in older men (n = 8) compared to young men (n = 7) and (2) if acute systemic infusion of ascorbic acid would enhance hypoxia-induced vasodilation in older men (n = 8). Heart rate, mean arterial pressure, oxygen saturation, minute ventilation, forearm vascular conductance (FVC, Doppler ultrasound), and cutaneous vascular conductance (CVC, laser Doppler flowmetry) were recorded continuously while subjects breathed 10% oxygen for 5 min. Changes from baseline were compared between groups and between treatments. The older adults had a significantly attenuated increase in FBF (13 ± 4 vs. 30 ± 7%) and FVC (16 ± 4 vs. 30 ± 7%) in response to 5 min of hypoxia. However, skin blood flow responses were comparable between groups (young: 35 ± 9, older: 30 ± 6%). In Experiment 2, FVC responses to 5 min of breathing 10% oxygen were not significantly different following saline (3 ± 10%) and ascorbic acid (8 ± 10%) in the older men. Ascorbic acid also had no physiological effects in the young men. These findings advance our basic understanding of how aging influences vascular responses to hypoxia and suggest that, in healthy humans, hypoxia-induced vasodilation is not restrained by reactive oxygen species. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  17. Crosstalk between nitric oxide and hypoxia-inducible factor signaling pathways: an update

    OpenAIRE

    Hendrickson MD; Poyton RO

    2015-01-01

    Marina D Hendrickson, Robert O Poyton Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA Abstract: Hypoxia-inducible factor-1 (HIF-1) is responsible for cellular adaptations to hypoxia. While oxygen (O2) negatively regulates its stability, many other factors affect HIF-1 stability and activity, including nitric oxide (NO). NO derived from l-arginine and nitrite (NO2–) could nitrosylate or nitrate HIF-1 and multiple proteins involv...

  18. The Protective Effect of Salidroside on Hypoxia-Induced Corpus Cavernosum Smooth Muscle Cell Phenotypic Transformation

    OpenAIRE

    Zhang, Xiang; Zhao, Jian-Feng; Zhao, Fan; Yan, Jun-Feng; Yang, Fan; Huang, Xiao-Jun; Chen, Gang; Fu, Hui-ying; Lv, Bo-Dong

    2017-01-01

    Salidroside, a major active ingredient isolated from Rhodiola rosea, has a long application in Chinese medical history. It has widely demonstrated effects on fatigue, psychological stress, and depression and exhibits potential antihypoxia activity. Emerging evidence shows that hypoxia is an important independent risk factor for erectile dysfunction (ED). The aim of this study was to clarify the effect of salidroside on hypoxia-induced phenotypic transformation of corpus cavernosum smooth musc...

  19. Global hypoxia induced impairment in learning and spatial memory is associated with precocious hippocampal aging.

    Science.gov (United States)

    Biswal, Suryanarayan; Sharma, Deepti; Kumar, Kushal; Nag, Tapas Chandra; Barhwal, Kalpana; Hota, Sunil Kumar; Kumar, Bhuvnesh

    2016-09-01

    Both chronological aging and chronic hypoxia stress have been reported to cause degeneration of hippocampal CA3 neurons and spatial memory impairment through independent pathways. However, the possible occurrence of precocious biological aging on exposure to single episode of global hypoxia resulting in impairment of learning and memory remains to be established. The present study thus aimed at bridging this gap in existing literature on hypoxia induced biological aging. Male Sprague Dawley rats were exposed to simulated hypobaric hypoxia (25,000ft) for different durations and were compared with aged rats. Behavioral studies in Morris Water Maze showed decline in learning abilities of both chronologically aged as well as hypoxic rats as evident from increased latency and pathlength to reach target platform. These behavioral changes in rats exposed to global hypoxia were associated with deposition of lipofuscin and ultrastructural changes in the mitochondria of hippocampal neurons that serve as hallmarks of aging. A single episode of chronic hypobaric hypoxia exposure also resulted in the up-regulation of pro-aging protein, S100A9 and down regulation of Tau, SNAP25, APOE and Sod2 in the hippocampus similar to that in aged rats indicating hypoxia induced accelerated aging. The present study therefore provides evidence for role of biological aging of hippocampal neurons in hypoxia induced impairment of learning and memory. Copyright © 2016. Published by Elsevier Inc.

  20. Hypoxia-induced deoxycytidine kinase contributes to epithelial proliferation in pulmonary fibrosis.

    Science.gov (United States)

    Weng, Tingting; Poth, Jens M; Karmouty-Quintana, Harry; Garcia-Morales, Luis J; Melicoff, Ernestina; Luo, Fayong; Chen, Ning-yuan; Evans, Christopher M; Bunge, Raquel R; Bruckner, Brian A; Loebe, Matthias; Volcik, Kelly A; Eltzschig, Holger K; Blackburn, Michael R

    2014-12-15

    Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with few therapeutic options. Apoptosis of alveolar epithelial cells, followed by abnormal tissue repair characterized by hyperplastic epithelial cell formation, is a pathogenic process that contributes to the progression of pulmonary fibrosis. However, the signaling pathways responsible for increased proliferation of epithelial cells remain poorly understood. To investigate the role of deoxycytidine kinase (DCK), an important enzyme for the salvage of deoxynucleotides, in the progression of pulmonary fibrosis. DCK expression was examined in the lungs of patients with IPF and mice exposed to bleomycin. The regulation of DCK expression by hypoxia was studied in vitro and the importance of DCK in experimental pulmonary fibrosis was examined using a DCK inhibitor and alveolar epithelial cell-specific knockout mice. DCK was elevated in hyperplastic alveolar epithelial cells of patients with IPF and in mice exposed to bleomycin. Increased DCK was localized to cells associated with hypoxia, and hypoxia directly induced DCK in alveolar epithelial cells in vitro. Hypoxia-induced DCK expression was abolished by silencing hypoxia-inducible factor 1α and treatment of bleomycin-exposed mice with a DCK inhibitor attenuated pulmonary fibrosis in association with decreased epithelial cell proliferation. Furthermore, DCK expression, and proliferation of epithelial cells and pulmonary fibrosis was attenuated in mice with conditional deletion of hypoxia-inducible factor 1α in the alveolar epithelium. Our findings suggest that the induction of DCK after hypoxia plays a role in the progression of pulmonary fibrosis by contributing to alveolar epithelial cell proliferation.

  1. Transcriptional control of adenosine signaling by hypoxia-inducible transcription factors during ischemic or inflammatory disease.

    Science.gov (United States)

    Poth, Jens M; Brodsky, Kelley; Ehrentraut, Heidi; Grenz, Almut; Eltzschig, Holger K

    2013-02-01

    Inflammatory lesions, ischemic tissues, or solid tumors are characterized by the occurrence of severe tissue hypoxia within the diseased tissue. Subsequent stabilization of hypoxia-inducible transcription factors-particularly of hypoxia-inducible factor 1α (HIF1A)--results in significant alterations of gene expression of resident cells or inflammatory cells that have been recruited into such lesions. Interestingly, studies of hypoxia-induced changes of gene expression identified a transcriptional program that promotes extracellular adenosine signaling. Adenosine is a signaling molecule that functions through the activation of four distinct adenosine receptors--the ADORA1, ADORA2A, ADORA2B, and ADORA3 receptors. Extracellular adenosine is predominantly derived from the phosphohydrolysis of precursor nucleotides, such as adenosine triphosphate or adenosine monophosphate. HIF1A-elicited alterations in gene expression enhance the enzymatic capacity within inflamed tissues to produce extracellular adenosine. Moreover, hypoxia-elicited induction of adenosine receptors--particularly of ADORA2B--results in increased signal transduction. Functional studies in genetic models for HIF1A or adenosine receptors implicate this pathway in an endogenous feedback loop that dampens excessive inflammation and promotes injury resolution, while at the same time enhancing ischemia tolerance. Therefore, pharmacological strategies to enhance HIF-elicited adenosine production or to promote adenosine signaling through adenosine receptors are being investigated for the treatment of acute inflammatory or ischemic diseases characterized by tissue hypoxia.

  2. Hypoxia-Inducible Regulation of a Prodrug-Activating Enzyme for Tumor-Specific Gene Therapy

    Directory of Open Access Journals (Sweden)

    Toru Shibata

    2002-01-01

    Full Text Available Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase. (20NTR gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT 1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% O2 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

  3. 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.

  4. Rapamycin: one drug, many effects

    Science.gov (United States)

    Li, Jing; Kim, Sang Gyun; Blenis, John

    2014-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth and metabolism. Deregulation of the mTOR pathway has been implicated in a number of human diseases such as cancer, diabetes, obesity, neurological diseases and genetic disorders. Rapamycin, a specific inhibitor of mTOR, has been shown to be useful in the treatment of certain diseases. Here we discuss its mechanism of action and highlight recent findings regarding the effects and limitations of rapamycin monotherapy and the potential utility of combination therapy with rapamycin. PMID:24508508

  5. Attenuation of pentylenetrazole-induced acute status epilepticus in rats by adenosine involves inhibition of the mammalian target of rapamycin pathway.

    Science.gov (United States)

    Wang, Yuliang; Liu, Xuewu; Wang, Yuan; Chen, Jinbo; Han, Tao; Su, Lei; Zang, Kejun

    2017-10-18

    Adenosine (ADO) has been characterized as an endogenous anticonvulsant and alternative therapeutic drug, but its mechanism is not entirely clear. This study aimed to examine the relationship of ADO with the mammalian target of rapamycin (mTOR) in a Wistar rat model of pentylenetetrazole (PTZ)-induced acute status epilepticus. ADO (200 mg/kg) was administered intraperitoneally 30 min before PTZ (55-65 mg/kg) treatment, and Western blot assays and immunohistochemistry were performed 3 h after the onset of acute status epilepticus to detect phospho-TOR and the downstream target of mTOR, phospho-S6. The expression of these phosphoproteins in the hippocampus was significantly increased in PTZ-treated rats, but this increase was attenuated by the addition of ADO. To further verify a role for ADO in attenuating mTOR activity, we also evaluated its ability to suppress mTOR activity in normal rats that were not treated with PTZ. Our results suggest that ADO suppresses mTOR and S6 phosphorylation in normal rats and that this suppression can be reversed by the application of Compound C, an inhibitor of AMP-activated protein kinase, which functions as an upstream suppressor of the mTOR pathway. Thus, our results provide a novel antiepileptic mechanism for ADO in suppressing mTOR pathway activation upon PTZ-induced acute status epilepticus.

  6. Rapamycin suppresses brain aging in senescence-accelerated OXYS rats.

    Science.gov (United States)

    Kolosova, Nataliya G; Vitovtov, Anton O; Muraleva, Natalia A; Akulov, Andrey E; Stefanova, Natalia A; Blagosklonny, Mikhail V

    2013-06-01

    Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span inC elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wister rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging.

  7. Tumor growth effects of rapamycin on human biliary tract cancer cells

    Directory of Open Access Journals (Sweden)

    Heuer Matthias

    2012-06-01

    Full Text Available Abstract Background Liver transplantation is an important treatment option for patients with liver-originated tumors including biliary tract carcinomas (BTCs. Post-transplant tumor recurrence remains a limiting factor for long-term survival. The mammalian target of rapamycin-targeting immunosuppressive drug rapamycin could be helpful in lowering BTC recurrence rates. Therein, we investigated the antiproliferative effect of rapamycin on BTC cells and compared it with standard immunosuppressants. Methods We investigated two human BTC cell lines. We performed cell cycle and proliferation analyses after treatment with different doses of rapamycin and the standard immunosuppressants, cyclosporine A and tacrolimus. Results Rapamycin inhibited the growth of two BTC cell lines in vitro. By contrast, an increase in cell growth was observed among the cells treated with the standard immunosuppressants. Conclusions These results support the hypothesis that rapamycin inhibits BTC cell proliferation and thus might be the preferred immunosuppressant for patients after a liver transplantation because of BTC.

  8. Hypoxia-inducible factor-1α polymorphisms and TSC1/2 mutations are complementary in head and neck cancers

    Directory of Open Access Journals (Sweden)

    Nikitakis Nikolaos G

    2006-01-01

    Full Text Available Abstract Background Polymorphisms or mutations in hypoxia inducible factor-1 alpha (HIF-1alpha that increases its activity and stability under normoxia have recently been identified. Likewise, disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 has been shown to result in abnormal accumulation of HIF-1α. Here, we investigate the novel polymorphisms in exon 12, that approximate the oxygen-dependent degradation domain of HIF-1alpha in five cell lines and 28 patients with oral squamous carcinomas. Moreover, we assess for the presence of polymorphisms and mutations in TSC1 and TSC2, to ascertain if dysregulation of such might complement HIF-1alpha expression. Results Denaturing high pressure liquid chromatography (DHPLC analysis on PCR fragments in exon 12 of HIF-1alpha from 28 patients with OSCC revealed that 6 of 28 patients had mismatched heteroduplex patterns. Genomic DNA was extracted from peripheral blood leukocytes and direct sequencing showed that in 5 of the six cases these changes represented polymorphisms while, one case was a somatic mutation. Analyses of TSC1 and TSC2 revealed heteroduplexes in exons: TSC1 exon 17; TSC2 exons 36,40, and 41. The relative levels of HIF-1alpha were significantly greater for tumors possessing a HIF-1alpha polymorphism or mutation within exon 12, whereas tumors possessing a deletion or polymorphism in TSC1/TSC2 displayed a trend for higher levels of HIF-1alpha. Western blot analyses for HIF-1alpha, TSC1 and TSC2 in five SCC cell lines revealed high levels of HIF-1alpha in SCC cells possessing TSC1 and/or TSC2 mutations. Wild-type TSC2 cells targeted with siRNA to TSC2 exhibited increased levels of HIF-1alpha. Transfection of a HIF-1alpha mutant produced higher levels of HIF-1alpha in TSC1/TSC2 mutant cell lines than in wild type cells. TSC1/TSC2 mutant cell lines administered Rapamycin blocked S6 phorphorylation and diminished the levels of HIF-1alpha to those observed in cell lines with wild

  9. Mechanism of hypoxia-induced NF-kappaB.

    Science.gov (United States)

    Culver, Carolyn; Sundqvist, Anders; Mudie, Sharon; Melvin, Andrew; Xirodimas, Dimitris; Rocha, Sonia

    2010-10-01

    NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease.

  10. Mechanism of Hypoxia-Induced NF-κB▿ †

    Science.gov (United States)

    Culver, Carolyn; Sundqvist, Anders; Mudie, Sharon; Melvin, Andrew; Xirodimas, Dimitris; Rocha, Sonia

    2010-01-01

    NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease. PMID:20696840

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

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    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.

  12. Tariquidar sensitizes multiple myeloma cells to proteasome inhibitors via reduction of hypoxia-induced P-gp-mediated drug resistance.

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    Muz, Barbara; Kusdono, Hubert D; Azab, Feda; de la Puente, Pilar; Federico, Cinzia; Fiala, Mark; Vij, Ravi; Salama, Noha N; Azab, Abdel Kareem

    2017-12-01

    Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

  13. The function of hypoxia-inducible factor (HIF is independent of the endoplasmic reticulum protein OS-9.

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    Ulf Brockmeier

    Full Text Available The protein "amplified in osteosarcoma-9" (OS-9 has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF, a transcription factor that adapts cells to insufficient oxygen supply (hypoxia. A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.

  14. Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

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    Okumura, Fumihiko; Joo-Okumura, Akiko; Nakatsukasa, Kunio; Kamura, Takumi

    2017-01-01

    Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways. Both HIF-α and B-Myb are targets of pVHL-mediated polyubiquitination and proteasomal degradation. Here, we show that knockdown of HIF-2α induces downregulation of B-Myb in 786-O cells, which are deficient in pVHL, and this downregulation is prevented by proteasome inhibition. In the presence of pVHL and under hypoxia-like conditions, B-Myb and HIF-2α are both upregulated, and the upregulation of B-Myb requires expression of HIF-2α. We also show that HIF-2α and B-Myb interact in the nucleus, and this interaction is mediated by the central region of HIF-2α and the C-terminal region of B-Myb. These data indicate that oncogenic HIF-2α stabilizes B-Myb to suppress VHL pathogenesis.

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

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    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.

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

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    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.

  17. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

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    Wang, Ronghai [Department of Urology, Linzi District People' s Hospital, Zibo, 255400 (China); Zhang, Ping, E-mail: zpskx001@163.com [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Li, Jinhang [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Guan, Hongzai [Laboratory Department, School of Medicine, Qingdao University, Qingdao, 266071 (China); Shi, Guangjun, E-mail: qdmhshigj@yahoo.com [Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao, 266071 (China)

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  18. Prenatal Hypoxia Induced Dysfunction in Cerebral Arteries of Offspring Rats.

    Science.gov (United States)

    Tang, Jiaqi; Li, Na; Chen, Xueyi; Gao, Qinqin; Zhou, Xiuwen; Zhang, Yingying; Liu, Bailin; Sun, Miao; Xu, Zhice

    2017-10-03

    Hypoxia during pregnancy could cause abnormal development and lead to increased risks of vascular diseases in adults. This study determined angiotensin II (AII)-mediated vascular dysfunction in offspring middle cerebral arteries (MCA). Pregnant rats were subjected to hypoxia. Vascular tension in offspring MCA by AII with or without inhibitors, calcium channel activities, and endoplasmic reticulum calcium stores were tested. Whole-cell patch clamping was used to investigate voltage-dependent calcium channel currents. mRNA expression was tested using quantitative real-time polymerase chain reaction. AII-mediated MCA constriction was greater in male offspring exposed to prenatal hypoxia. AT1 and AT2 receptors were involved in the altered AII-mediated vasoconstriction. Prenatal hypoxia increased baseline activities of L-type calcium channel currents in MCA smooth muscle cells. However, calcium currents stimulated by AII were not significantly changed, whereas nifedipine inhibited AII-mediated vasoconstrictions in the MCA. Activities of IP3/ryanodine receptor-operated calcium channels, endoplasmic reticulum calcium stores, and sarcoendoplasmic reticulum membrane Ca(2+)-ATPase were increased. Prenatal hypoxia also caused dysfunction of vasodilatation via the endothelium NO synthase. The mRNA expressions of AT1A, AT1B, AT2R, Cav1.2α1C, Cav3.2α1H, and ryanodine receptor RyR2 were increased in the prenatal-hypoxia group. Hypoxia in pregnancy could induce dysfunction in both contraction and dilation in the offspring MCA. AII-increased constriction in the prenatal-hypoxia group was not mainly dependent on the L-type and T-type calcium channels; it might predominantly rely on the AII receptors, IP3/ryanodine receptors, and the endoplasmic reticulum calcium store as well as calcium ATPase. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  19. Biphasic Rapamycin Effects in Lymphoma and Carcinoma Treatment.

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    Liu, Yang; Pandeswara, Srilakshmi; Dao, Vinh; Padrón, Álvaro; Drerup, Justin M; Lao, Shunhua; Liu, Aijie; Hurez, Vincent; Curiel, Tyler J

    2017-01-15

    mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L(+) CD8(+) central memory T cells in EL4 challenge, but without clinical benefit. LD rapamycin significantly enhanced DD treatment efficacy, but DD plus LD rapamycin treatment effects were independent of antitumor immunity. Instead, rapamycin upregulated EL4 IL2 receptor in vitro and in vivo, facilitating direct DD tumor cell killing. LD rapamycin augmented DD efficacy against B16 melanoma and a human B-cell lymphoma, but not against human Jurkat T-cell lymphoma or ID8agg ovarian cancer cells. Treatment effects correlated with IL2R expression, but mechanisms in some tumors were not fully defined. Overall, our data define a distinct, biphasic mechanisms of action of mTOR inhibition at doses that are clinically exploitable, including in T-cell lymphomas. Cancer Res; 77(2); 520-31. ©2016 AACR. ©2016 American Association for Cancer Research.

  20. Tomato FK506 Binding Protein 12KD (FKBP12 mediates the interaction between rapamycin and Target of Rapamycin (TOR

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    Fangjie Xiong

    2016-11-01

    Full Text Available Target of Rapamycin (TOR signaling is an important regulator in multiple organisms including yeast, plants and animals. However, the TOR signaling in plants is much less understood as compared to that in yeast and animals. TOR kinase can be efficiently suppressed by rapamycin in the presence of functional FK506 Binding Protein 12KD (FKBP12 in yeast and animals. In most examined higher plants rapamycin fails to inhibit TOR kinase due to the non-functional FKBP12. Here we find that tomato plants showed obvious growth inhibition when treated with rapamycin and the inhibitory phenotype is similar to suppression of TOR causing by active-site TOR inhibitors (asTORis such as KU63794, AZD8055 and Torin1. The chemical genetic assays using TOR inhibitors and heterologous expressing SlFKBP12 in Arabidopsis indicated that the TOR signaling is functional in tomato. The protein gel shifting and TOR inhibitors combination assays showed that SlFKBP12 can mediate the interaction between rapamycin and TOR. Furthermore, comparative expression profiling analysis between treatments with rapamycin and KU63794 identified highly overlapped Differentially Expressed Genes (DEGs which are involved in many anabolic and catabolic processes, such as photosynthesis, cell wall restructuring, and senescence in tomato. These observations suggest that SlFFBP12 is functional in tomato. The results provided basic information of TOR signaling in tomato, and also some new insights into how TOR controls plant growth and development through reprogramming the transcription profiles

  1. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair.

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    Pengzhen Wang

    Full Text Available Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1 and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic

  2. Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats

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    Hales Charles A

    2011-02-01

    Full Text Available Abstract Background CXCR4 is the receptor for chemokine CXCL12 and reportedly plays an important role in systemic vascular repair and remodeling, but the role of CXCR4 in development of pulmonary hypertension and vascular remodeling has not been fully understood. Methods In this study we investigated the role of CXCR4 in the development of pulmonary hypertension and vascular remodeling by using a CXCR4 inhibitor AMD3100 and by electroporation of CXCR4 shRNA into bone marrow cells and then transplantation of the bone marrow cells into rats. Results We found that the CXCR4 inhibitor significantly decreased chronic hypoxia-induced pulmonary hypertension and vascular remodeling in rats and, most importantly, we found that the rats that were transplanted with the bone marrow cells electroporated with CXCR4 shRNA had significantly lower mean pulmonary pressure (mPAP, ratio of right ventricular weight to left ventricular plus septal weight (RV/(LV+S and wall thickness of pulmonary artery induced by chronic hypoxia as compared with control rats. Conclusions The hypothesis that CXCR4 is critical in hypoxic pulmonary hypertension in rats has been demonstrated. The present study not only has shown an inhibitory effect caused by systemic inhibition of CXCR4 activity on pulmonary hypertension, but more importantly also has revealed that specific inhibition of the CXCR4 in bone marrow cells can reduce pulmonary hypertension and vascular remodeling via decreasing bone marrow derived cell recruitment to the lung in hypoxia. This study suggests a novel therapeutic approach for pulmonary hypertension by inhibiting bone marrow derived cell recruitment.

  3. Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells.

    Science.gov (United States)

    Eguchi, Ryoji; Toné, Shigenobu; Suzuki, Akio; Fujimori, Yoshihiro; Nakano, Takashi; Kaji, Kazuhiko; Ohta, Toshiro

    2009-01-15

    We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.

  4. Quantifying circulating hypoxia-induced RNA transcripts in maternal blood to determine in utero fetal hypoxic status.

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    Whitehead, Clare; Teh, Wan Tinn; Walker, Susan P; Leung, Cheryl; Mendis, Sonali; Larmour, Luke; Tong, Stephen

    2013-12-09

    Hypoxia in utero can lead to stillbirth and severe perinatal injury. While current prenatal tests can identify fetuses that are hypoxic, none can determine the severity of hypoxia/acidemia. We hypothesized a hypoxic/acidemic fetus would up-regulate and release hypoxia-induced mRNA from the fetoplacental unit into the maternal circulation, where they can be sampled and quantified. Furthermore, we hypothesized the abundance of hypoxia induced mRNA in the maternal circulation would correlate with severity of fetal hypoxia/acidemia in utero. We therefore examined whether abundance of hypoxia-induced mRNA in the maternal circulation correlates with the degree of fetal hypoxia in utero. We performed a prospective study of two cohorts: 1) longitudinal study of pregnant women undergoing an induction of labor (labor induces acute fetal hypoxia) and 2) pregnancies complicated by severe preterm growth restriction (chronic fetal hypoxia). For each cohort, we correlated hypoxia induced mRNA in the maternal blood with degree of fetal hypoxia during its final moments in utero, evidenced by umbilical artery pH or lactate levels obtained at birth. Gestational tissues and maternal bloods were sampled and mRNAs quantified by microarray and RT-PCR. Hypoxia-induced mRNAs in maternal blood rose across labor, an event that induces acute fetal hypoxia. They exhibited a precipitous increase across the second stage of labor, a particularly hypoxic event. Importantly, a hypoxia gene score (sum of the relative expression of four hypoxia-induced genes) strongly correlated with fetal acidemia at birth. Hypoxia-induced mRNAs were also increased in the blood of women carrying severely growth restricted preterm fetuses, a condition of chronic fetal hypoxia. The hypoxia gene score correlated with the severity of ultrasound Doppler velocimetry abnormalities in fetal vessels. Importantly, the hypoxia gene score (derived from mRNA abundance in maternal blood) was significantly correlated with the

  5. Rapamycin drives selection against a pathogenic heteroplasmic mitochondrial DNA mutation.

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    Dai, Ying; Zheng, Kangni; Clark, Joanne; Swerdlow, Russell H; Pulst, Stefan M; Sutton, James P; Shinobu, Leslie A; Simon, David K

    2014-02-01

    Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial disorders for which effective treatments are lacking. Emerging data indicate that selective mitochondrial degradation through autophagy (mitophagy) plays a critical role in mitochondrial quality control. Inhibition of mammalian target of rapamycin (mTOR) kinase activity can activate mitophagy. To test the hypothesis that enhancing mitophagy would drive selection against dysfunctional mitochondria harboring higher levels of mutations, thereby decreasing mutation levels over time, we examined the impact of rapamycin on mutation levels in a human cytoplasmic hybrid (cybrid) cell line expressing a heteroplasmic mtDNA G11778A mutation, the most common cause of Leber's hereditary optic neuropathy. Inhibition of mTORC1/S6 kinase signaling by rapamycin induced colocalization of mitochondria with autophagosomes, and resulted in a striking progressive decrease in levels of the G11778A mutation and partial restoration of ATP levels. Rapamycin-induced upregulation of mitophagy was confirmed by electron microscopic evidence of increased autophagic vacuoles containing mitochondria-like organelles. The decreased mutational burden was not due to rapamycin-induced cell death or mtDNA depletion, as there was no significant difference in cytotoxicity/apoptosis or mtDNA copy number between rapamycin and vehicle-treated cells. These data demonstrate the potential for pharmacological inhibition of mTOR kinase activity to activate mitophagy as a strategy to drive selection against a heteroplasmic mtDNA G11778A mutation and raise the exciting possibility that rapamycin may have therapeutic potential for the treatment of mitochondrial disorders associated with heteroplasmic mtDNA mutations, although further studies are needed to determine if a similar strategy will be effective for other mutations and other cell types.

  6. Differential sensitivity of oxidative and glycolytic muscles to hypoxia-induced muscle atrophy.

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    de Theije, C C; Langen, R C J; Lamers, W H; Gosker, H R; Schols, A M W J; Köhler, S E

    2015-01-15

    Hypoxia as a consequence of acute and chronic respiratory disease has been associated with muscle atrophy. This study investigated the sensitivity of oxidative and glycolytic muscles to hypoxia-induced muscle atrophy. Male mice were exposed to 8% normobaric oxygen for up to 21 days. Oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles were isolated, weighed, and assayed for expression profiles of the ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and glucocorticoid receptor (GR) and hypoxia-inducible factor-1α (HIF1α) signaling. Fiber-type composition and the capillary network were investigated. Hypoxia-induced muscle atrophy was more prominent in the EDL than the soleus muscle. Although increased expression of HIF1α target genes showed that both muscle types sensed hypoxia, their adaptive responses differed. Atrophy consistently involved a hypoxia-specific effect (i.e., not attributable to a hypoxia-mediated reduction of food intake) in the EDL only. Hypoxia-specific activation of the UPS and ALP and increased expression of the glucocorticoid receptor (Gr) and its target genes were also mainly observed in the EDL. In the soleus, stimulation of gene expression of those pathways could be mimicked to a large extent by food restriction alone. Hypoxia increased the number of capillary contacts per fiber cross-sectional area in both muscles. In the EDL, this was due to type II fiber atrophy, whereas in the soleus the absolute number of capillary contacts increased. These responses represent two distinct modes to improve oxygen supply to muscle fibers, but may aggravate muscle atrophy in chronic obstructive pulmonary disease patients who have a predominance of type II fibers. Copyright © 2015 the American Physiological Society.

  7. Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease.

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    Olson, Nels; van der Vliet, Albert

    2011-08-01

    Induction and activation of nitric oxide (NO) synthases (NOS) and excessive production of NO are common features of almost all diseases associated with infection and acute or chronic inflammation, although the contribution of NO to the pathophysiology of these diseases is highly multifactorial and often still a matter of controversy. Because of its direct impact on tissue oxygenation and cellular oxygen (O(2)) consumption and re-distribution, the ability of NO to regulate various aspects of hypoxia-induced signaling has received widespread attention. Conditions of tissue hypoxia and the activation of hypoxia-inducible factors (HIF) have been implicated in hypoxia or in cancer biology, but are also being increasingly recognized as important features of acute and chronic inflammation. Thus, the activation of HIF transcription factors has been increasingly implicated in inflammatory diseases, and recent studies have indicated its critical importance in regulating phagocyte function, inflammatory mediator production, and regulation of epithelial integrity and repair processes. Finally, HIF also appears to contribute to important features of tissue fibrosis and epithelial-to-mesenchymal transition, processes that are associated with tissue remodeling in various non-malignant chronic inflammatory disorders. In this review, we briefly summarize the current state of knowledge with respect to the general mechanisms involved in HIF regulation and the impact of NO on HIF activation. Secondly, we will summarize the major recent findings demonstrating a role for HIF signaling in infection, inflammation, and tissue repair and remodeling, and will address the involvement of NO. The growing interest in hypoxia-induced signaling and its relation with NO biology is expected to lead to further insights into the complex roles of NO in acute or chronic inflammatory diseases and may point to the importance of HIF signaling as key feature of NO-mediated events during these disorders

  8. L-arginine Attenuates Hypobaric Hypoxia-Induced Increase in Ornithine Decarboxylase 1.

    Science.gov (United States)

    Yuhong, Li; Zhengzhong, Bai; Feng, Tang; Quanyu, Yang; Ge, Ri-Li

    2017-12-01

    Chronic hypoxia-induced pulmonary hypertension and vascular remodeling have been shown to be associated with ornithine decarboxylase 1 (ODC1). However, few animal studies have investigated the role of ODC1 in acute hypoxia. We investigated ODC1 gene expression, morphologic and functional changes, and the effect of L-arginine as an attenuator in lung tissues of rats exposed to acute hypobaric hypoxia at a simulated altitude of 6000 m. Sprague-Dawley rats exposed to simulated hypobaric hypoxia (6000 m) for 24, 48, or 72 hours were treated with L-arginine (L-arginine group, 20 mg/100 g intraperitoneal; n=15) or untreated (non-L-arginine group, n=15). Control rats (n=5) were maintained at 2260 m in a normal environment for the same amount of time but were treated without L-arginine. The mean pulmonary artery pressure was measured by PowerLab system. The morphologic and immunohistochemical changes in lung tissue were observed under a microscope. The mRNA and protein levels of ODC1 were measured by real-time polymerase chain reaction and Western-blot, respectively. Hypobaric hypoxia induced pulmonary interstitial hyperemia and capillary expansion in the lungs of rats exposed to acute hypoxia at 6000 m. The mean pulmonary artery pressure and the mRNA and protein levels of ODC1 were significantly increased, which could be attenuated by treatment with L-arginine. L-arginine attenuates acute hypobaric hypoxia-induced increase in mean pulmonary artery pressure and ODC1 gene expression in lung tissues of rats. ODC1 gene contributes to the development of hypoxic pulmonary hypertension. Copyright © 2017. Published by Elsevier Inc.

  9. Iptakalim attenuates hypoxia-induced pulmonary arterial hypertension in rats by endothelial function protection.

    Science.gov (United States)

    Zhu, Rong; Bi, Li-Qing; Wu, Su-Ling; Li, Lan; Kong, Hui; Xie, Wei-Ping; Wang, Hong; Meng, Zi-Li

    2015-08-01

    The present study aimed to investigate the protective effects of iptakalim, an adenosine triphosphate (ATP)-sensitive potassium channel opener, on the inflammation of the pulmonary artery and endothelial cell injury in a hypoxia-induced pulmonary arterial hypertension (PAH) rat model. Ninety-six Sprague-Dawley rats were placed into normobaric hypoxia chambers for four weeks and were treated with iptakalim (1.5 mg/kg/day) or saline for 28 days. The right ventricle systolic pressures (RVSP) were measured and small pulmonary arterial morphological alterations were analyzed with hematoxylin and eosin staining. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the content of interleukin (IL)-1β and IL-10. Immunohistochemical analysis for ED1(+) monocytes was performed to detect the inflammatory cells surrounding the pulmonary arterioles. Western blot analysis was performed to analyze the expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and endothelial nitric oxide synthase (eNOS) in the lung tissue. Alterations in small pulmonary arteriole morphology and the ultrastructure of pulmonary arterial endothelial cells were observed via light and transmission electron microscopy, respectively. Iptakalim significantly attenuated the increase in mean pulmonary artery pressure, RVSP, right ventricle to left ventricle plus septum ratio and small pulmonary artery wall remodeling in hypoxia-induced PAH rats. Iptakalim also prevented an increase in IL-1β and a decrease in IL-10 in the peripheral blood and lung tissue, and alleviated inflammatory cell infiltration in hypoxia-induced PAH rats. Furthermore, iptakalim enhanced PECAM-1 and eNOS expression and prevented the endothelial cell injury induced by hypoxic stimuli. Iptakalim suppressed the pulmonary arteriole and systemic inflammatory responses and protected against the endothelial damage associated with the upregulation of PECAM-1 and eNOS, suggesting that iptakalim may represent a

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

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    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.

  11. Hesperidin induces apoptosis and triggers autophagic markers through inhibition of Aurora-A mediated phosphoinositide-3-kinase/Akt/mammalian target of rapamycin and glycogen synthase kinase-3 beta signalling cascades in experimental colon carcinogenesis.

    Science.gov (United States)

    Saiprasad, Gowrikumar; Chitra, Palanivel; Manikandan, Ramar; Sudhandiran, Ganapasam

    2014-09-01

    Abnormalities in the homeostasis mechanisms involved in cell survival and apoptosis are contributing factors for colon carcinogenesis. Interventions of these mechanisms by pharmacologically safer agents gain predominance in colon cancer prevention. We previously reported the chemopreventive efficacy of hesperidin against colon carcinogenesis. In the present study, we aimed at investigating the potential of hesperidin over the abrogated Aurora-A coupled pro-survival phosphoinositide-3-kinase (PI3K)/Akt signalling cascades. Further, the role of hesperidin over apoptosis and mammalian target of rapamycin (mTOR) mediated autophagic responses were studied. Azoxymethane (AOM) induced mouse model of colon carcinogenesis was involved in this study. Hesperidin treatment was provided either in initiation/post-initiation mode respectively. Hesperidin significantly altered AOM mediated anti-apoptotic scenario by modulating Bax/Bcl-2 ratio together with enhanced cytochrome-c release and caspase-3, 9 activations. In addition, hesperidin enhanced p53-p21 axis with concomitant decrease in cell cycle regulator. Hesperidin treatment caused significant up-regulation of tumour suppressor phosphatase and tensin homologue (PTEN) with a reduction in the expression of AOM mediated p-PI3K and p-Akt. Additionally, hesperidin administration exhibited inhibition against p-mTOR expression which in turn led to stimulation of autophagic markers Beclin-1 and LC3-II. Aurora-A an upstream regulator of PI3K/Akt pathway was significantly inhibited by hesperidin. Furthermore, hesperidin administration restored glycogen synthase kinase-3 beta (GSK-3β) activity which in turn prevented the accumulation of oncoproteins β-catenin, c-jun and c-myc. Taken together, hesperidin supplementation initiated apoptosis via targeted inhibition of constitutively activated Aurora-A mediated PI3K/Akt/GSK-3β and mTOR pathways coupled with autophagic stimulation against AOM induced colon carcinogenesis. Copyright

  12. Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

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    Goodwin, Justin; Choi, Hyunsung; Hsieh, Meng-Hsiung; Neugent, Michael L; Ahn, Jung-Mo; Hayenga, Heather N; Singh, Pankaj K; Shackelford, David B; Lee, In-Kyu; Shulaev, Vladimir; Dhar, Shanta; Takeda, Norihiko; Kim, Jung-Whan

    2018-02-01

    Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-β-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.

  13. Megakaryocytic leukemia 1 (MKL1 regulates hypoxia induced pulmonary hypertension in rats.

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    Zhibin Yuan

    Full Text Available Hypoxia induced pulmonary hypertension (HPH represents a complex pathology that involves active vascular remodeling, loss of vascular tone, enhanced pulmonary inflammation, and increased deposition of extracellular matrix proteins. Megakaryocytic leukemia 1 (MKL1 is a transcriptional regulator known to influence cellular response to stress signals in the vasculature. We report here that in response to chronic hypobaric hypoxia, MKL1 expression was up-regulated in the lungs in rats. Short hairpin RNA (shRNA mediated depletion of MKL1 significantly ameliorated the elevation of pulmonary arterial pressure in vivo with a marked alleviation of vascular remodeling. MKL1 silencing also restored the expression of NO, a key vasoactive molecule necessary for the maintenance of vascular tone. In addition, hypoxia induced pulmonary inflammation was dampened in the absence of MKL1 as evidenced by normalized levels of pro-inflammatory cytokines and chemokines as well as reduced infiltration of pro-inflammatory immune cells in the lungs. Of note, MKL1 knockdown attenuated fibrogenesis in the lungs as indicated by picrosirius red staining. Finally, we demonstrate that MKL1 mediated transcriptional activation of type I collagen genes in smooth muscle cells under hypoxic conditions. In conclusion, we data highlight a previously unidentified role for MKL1 in the pathogenesis of HPH and as such lay down groundwork for future investigation and drug development.

  14. Expression of hypoxia inducible factor 1 alpha and its clinical significance in esophageal carcinoma: A meta-analysis.

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    Jing, Shao Wu; Wang, Jun; Xu, Qing

    2017-07-01

    Many studies have analyzed the relationship between hypoxia inducible factor 1 alpha expression and its relation to differentiation, lymph node metastasis, and other clinicopathological variables of esophageal carcinoma, but the results are still inconsistent. This meta-analysis was carried out to explore hypoxia inducible factor 1 alpha in esophageal carcinoma and its correlation with clinicopathological features and prognosis, in order to provide comprehensive reference for clinic. A total of 18 studies including 1566 patients with esophageal squamous cell carcinoma were enrolled. The results showed that compared with para-carcinoma tissue, the expression of hypoxia inducible factor 1 alpha was significantly enhanced (odds ratio = 0.122, 95% confidence interval = 0.074-0.201, p = 0.000); hypoxia inducible factor 1 alpha was associated with differentiation (odds ratio = 1.458, 95% confidence interval = 1.108-1.920, p = 0.007), T classification (odds ratio = 0.457, 95% confidence interval = 0.265-0.786, p = 0.005), lymph node metastasis (odds ratio = 0.337, 95% confidence interval = 0.185-0.614, p = 0.000), and pathological tumor-node-metastasis stage (odds ratio = 0.362, 95% confidence interval = 0.177-0.740, p = 0.005), whereas there was no relation to histological grade, lymphatic vessel invasion, blood vessel invasion, 3- to 5-year overall survival and disease-free survival. Patients with hypoxia inducible factor 1 alpha overexpression had poor differentiation, increased depth of tumor invasion, more lymph node metastasis, and late pathological tumor-node-metastasis stage. Hypoxia inducible factor 1 alpha could be an indicator for differentiation, T classification, lymph node metastasis, and pathological tumor-node-metastasis stage, and it is worth further study.

  15. 3-Mercaptopyruvate Sulfurtransferase, Not Cystathionine β-Synthase Nor Cystathionine γ-Lyase, Mediates Hypoxia-Induced Migration of Vascular Endothelial Cells.

    Science.gov (United States)

    Tao, Beibei; Wang, Rui; Sun, Chen; Zhu, Yichun

    2017-01-01

    Hypoxia-induced angiogenesis is a common phenomenon in many physiological and patho-physiological processes. However, the potential differential roles of three hydrogen sulfide producing systems cystathionine γ-lyase (CSE)/H2S, cystathionine β-synthase (CBS)/H2S, and 3-mercaptopyruvate sulfurtransferase (MPST)/H2S in hypoxia-induced angiogenesis are still unknown. We found that minor hypoxia (10% oxygen) significantly increased the migration of vascular endothelial cells while hypoxia (8% oxygen) significantly inhibited cell migration. The present study was performed using cells cultured in 10% oxygen. RNA interference was used to block the endogenous generation of hydrogen sulfide by CSE, CBS, or MPST in a vascular endothelial cell migration model in both normoxia and hypoxia. The results showed that CBS had a promoting effect on the migration of vascular endothelial cells cultured in both normoxic and hypoxic conditions. In contrast, CSE had an inhibitory effect on cell migration. MPST had a promoting effect on the migration of vascular endothelial cells cultured in hypoxia; however, it had no effect on the cells cultured in normoxia. Importantly, it was found that the hypoxia-induced increase in vascular endothelial cell migration was mediated by MPST, but not CSE or CBS. The western blot analyses showed that hypoxia significantly increased MPST protein levels, decreased CSE protein levels and did not change CBS levels, suggesting that these three hydrogen sulfide-producing systems respond differently to hypoxic conditions. Interestingly, MPST protein levels were elevated by hypoxia in a bi-phasic manner and MPST mRNA levels increased later than the first stage elevation of the protein levels, implying that the expression of MPST induced by hypoxia was also regulated at a post-transcriptional level. RNA pull-down assay showed that some candidate RNA binding proteins, such as nucleolin and Annexin A2, were dissociated from the 3'-UTR of MPST mRNA in hypoxia

  16. Rapamycin: One Drug, Many Effects

    OpenAIRE

    Li, Jing; Kim, Sang Gyun; Blenis, John

    2014-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth and metabolism. Deregulation of the mTOR pathway has been implicated in a number of human diseases such as cancer, diabetes, obesity, neurological diseases and genetic disorders. Rapamycin, a specific inhibitor of mTOR, has been shown to be useful in the treatment of certain diseases. Here we discuss its mechanism of action and highlight recent findings regarding the effects and limitations of rapa...

  17. Small Molecule Inhibition of miR-544 Biogenesis Disrupts Adaptive Responses to Hypoxia by Modulating ATM-mTOR Signaling.

    Science.gov (United States)

    Haga, Christopher L; Velagapudi, Sai Pradeep; Strivelli, Jacqueline R; Yang, Wang-Yong; Disney, Matthew D; Phinney, Donald G

    2015-10-16

    Hypoxia induces a complex circuit of gene expression that drives tumor progression and increases drug resistance. Defining these changes allows for an understanding of how hypoxia alters tumor biology and informs design of lead therapeutics. We probed the role of microRNA-544 (miR-544), which silences mammalian target of rapamycin (mTOR), in a hypoxic breast cancer model by using a small molecule (1) that selectively impedes the microRNA's biogenesis. Application of 1 to hypoxic tumor cells selectively inhibited production of the mature microRNA, sensitized cells to 5-fluorouracil, and derepressed mRNAs affected by miR-544 in cellulo and in vivo, including boosting mTOR expression. Thus, small molecule inhibition of miR-544 reverses a tumor cell's physiological response to hypoxia. Importantly, 1 sensitized tumor cells to hypoxia-associated apoptosis at a 25-fold lower concentration than a 2'-O-methyl RNA antagomir and was as selective. Further, the apoptotic effect of 1 was suppressed by treatment of cell with rapamycin, a well-known inhibitor of the mTOR signaling pathway, illustrating the selectivity of the compound. Thus, RNA-directed chemical probes, which could also serve as lead therapeutics, enable interrogation of complex cellular networks in cells and animals.

  18. Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis

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    Jérôme eDoyen

    2013-01-01

    Full Text Available The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases CAIX and CAXII constitute a robust pHi-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX and LS174Tr cells (inducible knock-down for ca9/ca12 were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pHo manipulations and hypoxia (1% O2 exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pHi-regulating capacity of fibroblasts through inhibition of NHE-1 sensitize cells to radiation-induced cell death. Secondly, the pHi-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50% and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pHi regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pHi-regulating carbonic anhydrases as an anti-cancer strategy.

  19. Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

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    Kristy Zera

    Full Text Available Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

  20. Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

    Science.gov (United States)

    Zera, Kristy; Zastre, Jason

    2017-01-01

    Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD) is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

  1. Hypoxia-Induced Intrauterine Growth Restriction Increases the Susceptibility of Rats to High-Fat Diet–Induced Metabolic Syndrome

    Science.gov (United States)

    Rueda-Clausen, Christian F.; Dolinsky, Vernon W.; Morton, Jude S.; Proctor, Spencer D.; Dyck, Jason R.B.; Davidge, Sandra T.

    2011-01-01

    OBJECTIVE It is recognized that there is a remarkable variability in the systemic response to high-fat (HF) diets that cannot be completely explained by genetic factors. In addition, pregnancy complications leading to intrauterine growth restriction (IUGR) have been associated with an increased risk of developing metabolic syndrome (MetS) later in life. Thus, we hypothesized that offspring born with IUGR exhibit permanent metabolic changes that make them more susceptible to HF diet–induced MetS. RESEARCH DESIGN AND METHODS SD rats born normal (control) or with hypoxia-induced IUGR were randomized to low-fat (10% fat) or HF (45% fat) diets. After 9 weeks of feeding, physiological and molecular pathways involved in the MetS were evaluated. RESULTS IUGR offspring exhibited decreased energy intake and physical activity relative to controls. In offspring fed a HF diet, IUGR was associated with decreased total body fat content, a relative increase in intra-abdominal fat deposition and adipocyte size, an increase in fasting plasma concentrations of leptin, triglyceride and free fatty acids, and an increased concentration of triglycerides and ceramides in both liver and skeletal muscle. These changes in lipid homeostasis were accompanied by in vivo insulin resistance and impaired glucose tolerance and associated with increased phosphorylation of protein kinase C θ, inhibition of insulin receptor substrate 1, and a decreased activation of protein kinase B (PKB; also known as Akt) in liver and skeletal muscle in response to insulin. CONCLUSIONS IUGR enhances specific deleterious metabolic responses to a HF diet. Our results suggest that offspring born with IUGR may require special attention and follow-up to prevent the early onset of MetS. PMID:21270262

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

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    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.

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

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    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.

  4. Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells.

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    Veronica Kalhori

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1 is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3, Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.

  5. Evidence for a lack of a direct transcriptional suppression of the iron regulatory peptide hepcidin by hypoxia-inducible factors.

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    Melanie Volke

    2009-11-01

    Full Text Available Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1alpha or HIF-2alpha knock-down or by depletion of the HIF and iron regulatory protein (IRP target transferrin receptor 1 (TfR1. However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2, one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3 kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.

  6. Prolyl-hydroxylase inhibitor activating hypoxia-inducible transcription factors reduce levels of transplant arteriosclerosis in a murine aortic allograft model.

    Science.gov (United States)

    Heim, Christian; Bernhardt, Wanja; Jalilova, Sabina; Wang, Zhendi; Motsch, Benjamin; Ramsperger-Gleixner, Martina; Burzlaff, Nicolai; Weyand, Michael; Eckardt, Kai-Uwe; Ensminger, Stephan M

    2016-05-01

    The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection, is associated with a chronic immune response and also influenced by an initial injury to the graft through ischaemia and reperfusion. Hypoxia-inducible transcription factor (HIF)-1 pathway signalling has a protective effect against ischaemia-reperfusion injury and has already been demonstrated to ameliorate allograft nephropathy in previous animal studies. Therefore, the aim of this study was to investigate the effect of stabilization of hypoxia-inducible transcription factors with a prolyl-hydroxylase domain (PHD) inhibitor on transplant arteriosclerosis in an experimental aortic allograft model. MHC-class I mismatched C.B10-H2(b)/LilMcdJ donor thoracic aortas were heterotopically transplanted into the abdominal aorta of BALB/c mice. Donor animals received a single dose of the PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40 mg/kg) or vehicle i.p. 4 h before transplantation. Intragraft HIF accumulation after ICA treatment was detected by immunohistochemistry before and after cold ischaemia (n = 5). Grafts were harvested 30 days after transplantation and analysed by histology (n = 7) and immunofluorescence (n = 7). In addition, intragraft mRNA expression for cytokines, adhesion molecules and growth factors was determined on Day 14 (n = 7). Donor preconditioning with ICA resulted in HIF accumulation in the aorta and induction of the HIF target genes vascular endothelial growth factor and transforming growth factor-beta. Vascular lesions were present in both experimental groups. However, there was significantly reduced intimal proliferation in preconditioned grafts when compared with vehicle controls [intimal proliferation 31.3 ± 8% (ICA) vs 55.3 ± 20% (control), P arteriosclerosis and allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical

  7. mTORC1 inhibitors rapamycin and metformin affect cardiovascular markers differentially in ZDF rats.

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    Nistala, Ravi; Raja, Ahmad; Pulakat, Lakshmi

    2017-03-01

    Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.

  8. Hypoxia-Inducible Factor-1α Expression in Kidney Transplant Biopsy Specimens After Reperfusion Is Associated With Early Recovery of Graft Function After Cadaveric Kidney Transplantation.

    Science.gov (United States)

    Oda, T; Ishimura, T; Yokoyama, N; Ogawa, S; Miyake, H; Fujisaw, M

    Ischemia/reperfusion injury during kidney transplantation (KTx) delays allograft recovery. Hypoxia-inducible factor-1α (HIF-1α) is the key regulator of the protective response to ischemia/reperfusion injury. We evaluated the impact of the HIF-1α signaling pathway on allograft recovery during cadaveric KTx. Between 1996 and 2015, 46 patients underwent cadaveric KTx. The expression levels of HIF-1α-related proteins, including phosphoinositide 3-kinase, phosphorylated (p)-Akt, p-mammalian target of rapamycin, p-Eukaryotic translation initiation factor 4E, p-S6 ribosomal protein, and HIF-1α, were immunohistochemically evaluated and semi-quantitatively scored in graft biopsy specimens after 1 hour of revascularization. Ten kidney biopsy specimens collected during donor nephrectomy for living KTx were used as controls. Delayed graft function (DGF) was defined as the need for dialysis within 1 week of KTx. We compared the staining scores of each protein and several clinical parameters between patients with and those without DGF. Expression levels of all six proteins in specimens after revasculization were elevated compared with those in controls. Thirty-five patients had DGF. Expression levels of PI3K, p-AKT, p-mTOR, p-eIF4E, and HIF-1α were significantly higher in patients without DGF than in those with DGF. Univariate analysis identified expression levels of p-Akt, p-S6, and HIF-1α, in addition to donor type (heart beating/non-heart beating), cold ischemic time, and donor age as significant predictors of DGF. Of these, only expression levels of HIF-1α and donor type were independently associated with DGF in multivariate analysis. Up-regulation of HIF-1α in allografts after reperfusion may be a predictor of early recovery after cadaveric KTx. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Longitudinal imaging studies of tumor microenvironment in mice treated with the mTOR inhibitor rapamycin.

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    Keita Saito

    Full Text Available Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII by electron paramagnetic resonance imaging (EPRI and magnetic resonance imaging (MRI to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3 and measurements of tumor pO(2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2<10 mm Hg in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

  10. Reperfusion Therapy with Rapamycin Attenuates Myocardial Infarction through Activation of AKT and ERK

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    Scott M. Filippone

    2017-01-01

    Full Text Available Prompt coronary reperfusion is the gold standard for minimizing injury following acute myocardial infarction. Rapamycin, mammalian target of Rapamycin (mTOR inhibitor, exerts preconditioning-like cardioprotective effects against ischemia/reperfusion (I/R injury. We hypothesized that Rapamycin, given at the onset of reperfusion, reduces myocardial infarct size through modulation of mTOR complexes. Adult C57 male mice were subjected to 30 min of myocardial ischemia followed by reperfusion for 1 hour/24 hours. Rapamycin (0.25 mg/kg or DMSO (7.5% was injected intracardially at the onset of reperfusion. Post-I/R survival (87% and cardiac function (fractional shortening, FS: 28.63±3.01% were improved in Rapamycin-treated mice compared to DMSO (survival: 63%, FS: 17.4±2.6%. Rapamycin caused significant reduction in myocardial infarct size (IS: 26.2±2.2% and apoptosis (2.87±0.64% as compared to DMSO-treated mice (IS: 47.0±2.3%; apoptosis: 7.39±0.81%. Rapamycin induced phosphorylation of AKT S473 (target of mTORC2 but abolished ribosomal protein S6 phosphorylation (target of mTORC1 after I/R. Rapamycin induced phosphorylation of ERK1/2 but inhibited p38 phosphorylation. Infarct-limiting effect of Rapamycin was abolished with ERK inhibitor, PD98059. Rapamycin also attenuated Bax and increased Bcl-2/Bax ratio. These results suggest that reperfusion therapy with Rapamycin protects the heart against I/R injury by selective activation of mTORC2 and ERK with concurrent inhibition of mTORC1 and p38.

  11. Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast

    NARCIS (Netherlands)

    Kuijper, Arno; Groep, P. van der; Wall, E. van der; Diest, P.J. van

    2005-01-01

    INTRODUCTION Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance

  12. Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis

    NARCIS (Netherlands)

    Sluimer, Judith C.; Gasc, Jean-Marie; van Wanroij, Job L.; Kisters, Natasja; Groeneweg, Mathijs; Sollewijn Gelpke, Maarten D.; Cleutjens, Jack P.; van den Akker, Luc H.; Corvol, Pierre; Wouters, Bradly G.; Daemen, Mat J.; Bijnens, Ann-Pascale J.

    2008-01-01

    We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of

  13. Hypoxia Inducible Factor 3α Plays a Critical Role in Alveolarization and Distal Epithelial Cell Differentiation during Mouse Lung Development

    NARCIS (Netherlands)

    Y. Huang (Yao); J.K. Ochieng (Joshua); M. van Kempen (Marjon); A. Boerema-de Munck (Anne); S.M.A. Swagemakers (Sigrid); W.F.J. van IJcken (Wilfred); F.G. Grosveld (Frank); D. Tibboel (Dick); R.J. Rottier (Robbert)

    2013-01-01

    textabstractLung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α

  14. Current advances in the novel functions of hypoxia-inducible factor and prolyl hydroxylase in invertebrates.

    Science.gov (United States)

    Wang, L; Cui, S; Ma, L; Kong, L; Geng, X

    2015-12-01

    Oxygen is essential for aerobic life, and hypoxia has very severe consequences. Organisms need to overcome low oxygen levels to maintain biological functions during normal development and in disease states. The mechanism underlying the hypoxic response has been widely investigated in model animals such as Drosophila melanogaster and Caenorhabditis elegans. Hypoxia-inducible factor (HIF), a key gene product in the response to oxygen deprivation, is primarily regulated by prolyl hydroxylase domain enzymes (PHDs). However, recent findings have uncovered novel HIF-independent functions of PHDs. This review provides an overview of how invertebrates are able to sustain hypoxic damages, and highlights some recent discoveries in the regulation of cellular signalling by PHDs. Given that some core genes and major pathways are evolutionarily conserved, these research findings could provide insight into oxygen-sensitive signalling in mammals, and have biomedical implications for human diseases. © 2015 The Royal Entomological Society.

  15. Muscle-specific expression of hypoxia-inducible factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Mounier, Rémi; Pedersen, Bente Klarlund; Plomgaard, Peter

    2010-01-01

    fibres that possess unique patterns of protein and gene expression, producing different capillarization and energy metabolism systems. In this work, we analysed HIF-1alpha mRNA and protein expression related to the fibre-type composition in untrained human skeletal muscle by obtaining muscle biopsies......Skeletal muscle is well known to exhibit a high degree of plasticity depending on environmental changes, such as various oxygen concentrations. Studies of the oxygen-sensitive subunit alpha of hypoxia-inducible factor-1 (HIF-1) are difficult owing to the large variety of functionally diverse muscle......alpha mRNA and protein owing to their higher oxidative capacity. We have shown, in normoxic conditions, a higher HIF-1alpha protein expression in predominantly oxidative muscles than in predominantly glycolytic muscles. However, the HIF-1alpha mRNA expression pattern was not in agreement with the HIF-1...

  16. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Alsner, Jan; Overgaard, Jens

    2007-01-01

    BACKGROUND: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression......Ha and FaDu(DD) cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu(DD) cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu(DD). Glut1 and LDH-A had...

  17. Hypoxia-inducible factor-1α: a promising therapeutic target for autoimmune diseases.

    Science.gov (United States)

    Guan, Shi-Yang; Leng, Rui-Xue; Tao, Jin-Hui; Li, Xiang-Pei; Ye, Dong-Qing; Olsen, Nancy; Zheng, Song Guo; Pan, Hai-Feng

    2017-07-01

    Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in both innate and adaptive immunity. Emerging evidence indicates that HIF-1α is associated with the inflammation and pathologic activities of autoimmune diseases. Areas covered: Considering that the types of autoimmune diseases are complicated and various, this review aims to cover the typical kinds of autoimmune diseases, discuss the molecular mechanisms, biological functions and expression of HIF-1α in these diseases, and further explore its therapeutic potential. Expert opinion: Inflammation and hypoxia are interdependent. HIF-1α as a key regulator of hypoxia, exerts a crucial role in the balance between Th17 and Treg, and involves in the inflammation and pathologic activities of autoimmune diseases. Although there are many challenges remaining to be overcome, targeting HIF-1α could be a promising strategy for autoimmune diseases therapies.

  18. Rv1894c Is a Novel Hypoxia-Induced Nitronate Monooxygenase Required for Mycobacterium tuberculosis Virulence

    Science.gov (United States)

    Klinkenberg, Lee G.; Karakousis, Petros C.

    2013-01-01

    Tuberculosis is difficult to cure, requiring a minimum of 6 months of treatment with multiple antibiotics. Small numbers of organisms are able to tolerate the antibiotics and persist in the lungs of infected humans, but they still require some metabolic activity to survive. We studied the role of the hypoxia-induced Rv1894c gene in Mycobacterium tuberculosis virulence in guinea pigs, which develop hypoxic, necrotic granulomas histologically resembling those in humans and found this gene to be necessary for full bacillary growth and survival. We characterized the function of the encoded enzyme as a nitronate monooxygenase, which is needed to prevent the buildup of toxic products during hypoxic metabolism and is negatively regulated by the transcriptional repressor KstR. Future studies will focus on developing small-molecule inhibitors that target Rv1894c and its homologs, with the goal of killing persistent bacteria, thereby shortening the time needed to treat tuberculosis. PMID:23408846

  19. The role of hypoxia-inducible factor-2 in digestive system cancers.

    Science.gov (United States)

    Zhao, J; Du, F; Shen, G; Zheng, F; Xu, B

    2015-01-15

    Hypoxia is an all but ubiquitous phenomenon in cancers. Two known hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, primarily mediate the transcriptional response to hypoxia. Despite the high homology between HIF-1α and HIF-2α, emerging evidence suggests differences between both molecules in terms of transcriptional targets as well as impact on multiple physiological pathways and tumorigenesis. To date, much progress has been made toward understanding the roles of HIF-2α in digestive system cancers. Indeed, HIF-2α has been shown to regulate multiple aspects of digestive system cancers, including cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy. These findings make HIF-2α a critical regulator of this malignant phenotype. Here we summarize the function of HIF-2 during cancer development as well as its contribution to tumorigenesis in digestive system malignancies.

  20. Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications

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    So Yeon Kim

    2015-11-01

    Full Text Available Hypoxia-inducible factor (HIF prolyl hydroxylases (PHDs are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

  1. Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish

    DEFF Research Database (Denmark)

    Kamei, Hiroyasu; Lu, Ling; Jiao, Shuang

    2008-01-01

    Background: Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability...... to genetic and experimental manipulation and because it possess a large number of duplicated genes. Methodology/Principal Findings: We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1...... is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains...

  2. Hypoxia-inducible factor 1 alpha is a poor prognostic factor and potential therapeutic target in malignant peripheral nerve sheath tumor.

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    Suguru Fukushima

    Full Text Available Malignant peripheral nerve sheath tumor (MPNST is a rare soft tissue sarcoma with poor prognosis. Hypoxia-inducible factor 1 (HIF-1 plays a crucial role in the cellular response to hypoxia and regulates the expression of multiple genes involved in tumor progression in various cancers. However, the importance of the expression of HIF-1α in MPNSTs is unclear.The expression of HIF-1α was examined immunohistochemically in 82 MPNST specimens. Cell culture assays of human MPNST cells under normoxic and hypoxic conditions were used to evaluate the impact of anti-HIF-1α-specific siRNA inhibition on cell survival. A screening kit was employed to identify small molecules that inhibited HIF-1α.The nuclear expression of HIF-1α was positive in 75.6% of MPNST samples (62/82 cases. Positivity for HIF-1α was a significant poor prognostic factor both in univariate (P = 0.048 and multivariate (P ≤ 0.0001 analyses. HIF-1α knockdown abrogated MPNST cell growth, inducing apoptosis. Finally, chetomin, an inhibitor of HIF-1α, effectively inhibited the growth of MPNST cells and induced their apoptosis.Inhibition of HIF-1α signaling is a potential treatment option for MPNSTs.

  3. [Effect of hypoxia-inducible factor-1α, endothelin-1 and inducible nitric oxide synthase in the pathogenesis of hypoxia-induced pulmonary hypertension of the neonatal rats].

    Science.gov (United States)

    Sang, Kui; Zhou, Ying; Li, Ming-xia

    2012-12-01

    To study the effect of hypoxia-inducible factor-1α (HIF-1α) in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH) of the neonatal rats through the study on the expression level of HIF-1α and its regulation factors: endothelin-1 (ET-1) and inducible nitric oxide synthase (iNOS) in blood serum and lung tissue. To make an HPH model of neonatal rats, 120 newborn Wistar rats were divided at random into two groups: HPH group and the regular oxygen controlled group with the same birthday. The rats of the two groups were put in the condition of hypoxia for 3, 5, 7, 10, 14, 21 days and then 10 rats of HPH group and control group were picked up, their mean pulmonary arterial pressure (mPAP), serum HIF-1α, and iNOS, and ET-1 content were tested, and finally their lung tissue was taken after they were sacrificed and the expression level of the gene mRNA of HIF-1α, iNOS and ET-1. (1) The rats experienced hypoxia for 3, 5, 7, 10, 14 or 21 days had an increasing mPAP: [8.47 ± 1.45, 10.04 ± 1.69, 10.89 ± 2.97, 16.96 ± 1.97, 13.01 ± 1.93, 21.04 ± 2.13 (mm Hg)], which had a significant differences compared with control groups [5.11 ± 1.06, 8.12 ± 1.11, 8.77 ± 0.92, 12.23 ± 1.78, 8.89 ± 0.89, 11.09 ± 1.64 (mm Hg)] (P rats in hypoxia group had a higher serum HIF-1α [0.83 ± 0.07, 0.84 ± 0.17, 0.97 ± 0.13, 1.10 ± 0.30, 0.92 ± 0.19 (pg/nmol)] than the control group [0.26 ± 0.20, 0.37 ± 0.16, 0.44 ± 0.18, 0.41 ± 0.23, 0.66 ± 0.18 (pg/nmol)] as they experienced hypoxia for 3, 5, 7, 10, and 14 days (P 0.05), and the content of serum iNOS after hypoxia for 14 or 21 days (4.56 ± 0.96, 5.86 ± 1.76) µmol/L was lower than that of the control group (10.35 ± 1.99, 8.44 ± 2.76) µmol/L (P rats and causedn a imbalance of ET-1 and NO. HIF-1α, ET-1 and iNOS altogether contributed to the occurrence and development of HPH in neonatal rats.

  4. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334 ameliorates murine colitis

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    Gupta R

    2014-01-01

    Full Text Available Ram Gupta,1 Anita R Chaudhary,2 Binita N Shah,1 Avinash V Jadhav,3 Shitalkumar P Zambad,1 Ramesh Chandra Gupta,4 Shailesh Deshpande,4 Vijay Chauthaiwale,4 Chaitanya Dutt4 1Department of Pharmacology, 2Cellular and Molecular Biology, 3Preclinical Safety Evaluation, 4Discovery, Torrent Research Centre, Torrent Pharmaceuticals Ltd, Gandhinagar, Gujarat, India Background and aim: Mucosal healing in inflammatory bowel disease (IBD can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods: The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn's disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results: TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion: Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor

  5. 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.

  6. Protective effects of myricetin on acute hypoxia-induced exercise intolerance and mitochondrial impairments in rats.

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    Dan Zou

    Full Text Available Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro.Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM. The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA. mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence.Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regulators, in addition, AMP-activated protein kinase(AMPK plays a crucial role in this process.Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis.

  7. Role of hypoxia and hypoxia inducible factor in physiological and pathological conditions

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    Mozhgan Jahani

    2017-11-01

    Full Text Available Introduction: Organisms are exposed to oxygen deprivation (Hypoxia in various physiological and pathological conditions. There are different conserve evolutionary responses to counterview with this stress that primary transcriptional response to stress related to hypoxia is interceded by hypoxia-inducible factor (HIF-1 in mammals. This factor can regulate different genes that have essential roles in adaptation to this condition. In this review, the role of this factor in physiological and pathological conditions under hypoxic condition has been evaluated after examining structural features and regulation characteristics of HIF-1. Methods: First, articles related to the keywords of hypoxia and HIF-1 (from 1991-2016 were searched from valid databases such as Springer Link, Google Scholar, PubMed and Science direct. Then, the articles correlated with hypoxia, HIF-1 and their roles in physiological and pathological conditions (120 articles were searched and just 64 articles were selected for this study. Result: According to studies, there are different genes in cells and organs that can be regulated by HIF-1. Activation of genes expression by this protein occurs through its linkage to cis-acting of 50 base pair hypoxia response element (HRE region located in their promotor and enhancer. Depending on circumstances, activation of these genes can be beneficial or harmful. Conclusion: Activation of different genes in hypoxia by HIF-1 has different effects on physiological and pathological conditions. Therefore, HIF-1, as a hypoxia-inducible factor in hypoxic conditions, plays an essential role in the adaptation of cells and organs to changes related to the presence of oxygen.

  8. Suppression of Phosphatidylinositol 3-Kinase/Akt Signaling Attenuates Hypoxia-Induced Pulmonary Hypertension Through the Downregulation of Lysyl Oxidase.

    Science.gov (United States)

    Xia, Xiao-Dong; Lee, Jasmine; Khan, Sajid; Ye, Leping; Li, Yuan; Dong, Liang

    2016-10-01

    Lysyl oxidase (LOX) is a copper-dependent enzyme that catalyzes covalent cross-linking of collagen. In response to hypoxia, phosphatidylinositol 3-kinase (PI3K) pathway is activated and contributes to pulmonary arterial hypertension (PAH). However, potential role of LOX in hypoxia-induced PAH is poorly understood. In this study, we explored the mechanism responsible for the development of hypoxia-induced PAH. Potent inhibitors of PI3K/Akt and LOX, wortmannin and β-aminopropionitrile (β-APN), were administrated in rat model of hypoxia-induced PAH. The cross-linking of collagen was assessed by the determination of hydroxyproline. LOX, LOXL-1, LOXL-2, LOXL-3, LOXL-4, Akt, and phospho-Akt expression was detected by real-time polymerase chain reaction and western blot analysis. We observed that collagen cross-linking and LOX activity were elevated in hypoxia-exposed rat lung tissue, but these effects were reversed by β-APN and wortmannin. In addition, exposure to hypoxia enhanced mRNA and protein expression and activity of LOX and LOXL-1 in a PI3K/Akt-dependent manner and induced the development of PAH. After the administration of wortmannin, the upregulation of LOX and cross-linking of collagen were significantly reversed in hypoxia-exposed rat pulmonary artery tissue. Taken together, the present study demonstrated that the upregulation of LOX expression and collagen cross-linking is PI3K/Akt dependent in rat with hypoxia-induced PAH. Suppression of PI3K/Akt pathway may alleviate hypoxia-induced PAH through the downregulation of LOX.

  9. Beneficial role of rapamycin in experimental autoimmune myositis.

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    Nicolas Prevel

    Full Text Available We developed an experimental autoimmune myositis (EAM mouse model of polymyositis where we outlined the role of regulatory T (Treg cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin in vivo in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment, or during 10 days following the last myosin immunization (curative treatment.Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R(2 = -0.645, p<0.0001. Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9 ± 2.2% vs. 9.3 ± 1.4%, p<0.001, which were mostly activated regulatory T cells (CD62L(lowCD44(high: 58.1 ± 5.78% vs. 33.1 ± 7%, treated vs. untreated, p<0.001. In rapamycin treated mice, inhibition of proliferation (Ki-67(+ is more important in effector T cells compared to Tregs cells (p<0.05. Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44(low CD62L(high naive T cell and in CD62L(low CD44(high activated T cell.Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.

  10. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

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    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  11. Assessment of Response of Kidney Tumors to Rapamycin and Atorvastatin in Tsc1+/- Mice.

    Science.gov (United States)

    Shen, Ming Hong; Samsel, Paulina; Shen, Louise L; Narov, Kalin; Yang, Jian; Sampson, Julian R

    2017-10-01

    Atorvastatin is widely used to lower blood cholesterol and to reduce risk of cardiovascular disease-associated complications. Epidemiological investigations and preclinical studies suggest that statins such as atorvastatin have antitumor activity for various types of cancer. Tuberous sclerosis (TSC) is a tumor syndrome caused by TSC1 or TSC2 mutations that lead to aberrant activation of mTOR and tumor formation in multiple organs. Previous studies have demonstrated that atorvastatin selectively suppressed growth and proliferation of mouse Tsc2 null embryonic fibroblasts through inhibition of mTOR. However, atorvastatin alone did not reduce tumor burden in the liver and kidneys of Tsc2+/- mice as assessed by histological analysis, and no combination therapy of rapamycin and atorvastatin has been tried. In this study, we used T2-weighted magnetic resonance imaging to track changes in tumor number and size in the kidneys of a Tsc1+/- mouse model and to assess the efficacy of rapamycin and atorvastatin alone and as a combination therapy. We found that rapamycin alone or rapamycin combined with atorvastatin significantly reduced tumor burden, while atorvastatin alone did not. Combined therapy with rapamycin and atorvastatin appeared to be more effective for treating renal tumors than rapamycin alone, but the difference was not statistically significant. We conclude that combined therapy with rapamycin and atorvastatin is unlikely to provide additional benefit over rapamycin as a single agent in the treatment of Tsc-associated renal tumors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

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    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

  13. Paradoxical regulation of hypoxia inducible factor-1α (HIF-1α by histone deacetylase inhibitor in diffuse large B-cell lymphoma.

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    Savita Bhalla

    Full Text Available Hypoxia inducible factor (HIF is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI, enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

  14. Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells.

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    Golan, Maya; Mabjeesh, Nicola J

    2017-05-09

    Hypoxia-inducible factor 1 (HIF-1) is a major mediator of the hypoxic response involved in tumor progression. We had earlier described the interaction between septin 9 isoform 1 (SEPT9_i1) protein and the oxygen-regulated subunit, HIF-1α. SEPT9_i1 is a member of the conserved family of GTP-binding cytoskeleton septins. SEPT9_i1 stabilizes HIF-1α and facilitates its cytoplasmic-nuclear translocation. We utilized split yellow fluorescent protein (YFP) bimolecular fluorescence complementation (BiFC) methodology to monitor the interaction between HIF-1α and SEPT9_i1 in live cells. N-terminal (YN) and C-terminal (YC) split YFP chimeras with HIF-1α and SEPT9_i1 on both their amino and carboxyl termini were generated. HIF-1α and SEPT9_i1 chimeras were expressed in cancer cells and screened for functional complementation. SEPT9_i1-YN and YC-HIF-1α formed a long-lived highly stable complex upon interaction. The BiFC signal was increased in the presence of hypoxia-mimicking agents. In contrast, YC-ΔHLH-HIF-1α chimera, which lacked the helix-loop-helix domain that is essential for the interaction with SEPT9_i1 as well as the expression of SEPT9_i1 252-379 amino acids fragment required for the interaction with HIF-1α, significantly reduced the BiFC signal. The signal was also reduced when cells were treated with 17-N-allylamino-17-demethoxygeldanamycin, an HSP90 inhibitor that inhibits HIF-1α. It was increased with fourchlorfenuron, a small molecule that increases the interaction between HIF-1α and SEPT9_i1. These results reconfirmed the interaction between HIF-1α and SEPT9_i1 that was imaged in live cells. This BiFC system represents a novel approach for studying the real-time interaction between these two proteins and will allow high-throughput drug screening to identity compounds that disrupt this interaction.

  15. Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB.

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    Nadine Rohwer

    Full Text Available BACKGROUND: Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1alpha has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1alpha's role in determining chemosensitivity focussing on responsible molecular pathways. METHODOLOGY AND PRINCIPAL FINDINGS: RNA interference was applied to inactivate HIF-1alpha or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-kappaB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1alpha in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1alpha-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1alpha markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1alpha-competent cells was identified as the molecular mechanism of HIF-1alpha-mediated inhibition of p53. Furthermore, loss of HIF-1alpha abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-kappaB and expression of anti-apoptotic NF-kappaB target genes. Accordingly, reconstitution of the NF-kappaB subunit p65 reversed the increased chemosensitivity of

  16. The hypoxia-inducible factor-1? activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia

    OpenAIRE

    Zhang, Qian; Doucet, Michele; Tomlinson, Ryan E; Han, Xiaobin; Quarles, L Darryl; Collins, Michael T; Clemens, Thomas L

    2016-01-01

    Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome in which ectopic production of fibroblast growth factor 23 (FGF23) by non-malignant mesenchymal tumors causes phosphate wasting and bone fractures. Recent studies have implicated the hypoxia-inducible factor-1? (HIF-1?) in other phosphate wasting disorders caused by elevated FGF23, including X-linked hypophosphatemic rickets and autosomal dominant hypophosphatemia. Here we provide evidence that HIF-1? mediates aberrant FGF23 i...

  17. Potential use of rapamycin in HIV infection

    DEFF Research Database (Denmark)

    Donia, Marco; McCubrey, James A; Bendtzen, Klaus

    2010-01-01

    The strong need for the development of alternative anti-HIV agents is primarily due to the emergence of strain-resistant viruses, the need for sustained adherence to complex treatment regimens and the toxicity of currently used antiviral drugs. This review analyzes proof of concept studies...... indicating that the immunomodulatory drug rapamycin (RAPA) possesses anti-HIV properties both in vitro and in vivo that qualifies it as a potential new anti-HIV drug. It represents a literature review of published studies that evaluated the in vitro and in vivo activity of RAPA in HIV. RAPA represses HIV-1...... replication in vitro through different mechanisms including, but not limited, to down regulation of CCR5. In addition RAPA synergistically enhances the anti-HIV activity of entry inhibitors such as vicriviroc, aplaviroc and enfuvirtide in vitro. RAPA also inhibits HIV-1 infection in human peripheral blood...

  18. Crocin attenuates acute hypobaric hypoxia-induced cognitive deficits of rats.

    Science.gov (United States)

    Zhang, Xiao-Yan; Zhang, Xian-Jun; Xv, Jin; Jia, Wei; Pu, Xiao-Yan; Wang, Hai-Yan; Liang, Hong; Zhuoma-Lamao; Lu, Dian-Xiang

    2018-01-05

    This study investigated whether crocin exerted neuroprotective effects against acute hypobaric hypoxia at high altitude in vivo and determined the underlying mechanisms. Male Sprague-Dawley rats were randomly assigned to a normoxic group,a hypoxic group, and three crocin groups at three different doses. The rats were transferred from 50m to 4200m for 3 days after treatment with crocin for 3 days. The learning and memory of the rat were evaluated with the Morris water maze test. Transmission electron microscope (TEM) was used to analyze the changes in the ultrastructure of hippocampal neurons. Peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and sirtuin-1 (SIRT1) levels were determined using immunohistochemical staining and western blotting. The escape latency of the crocin group was shorter than that of the hypoxic group, while the frequency of the rats reaching the platform was significantly higher in the crocin group. The structures of nerve cells and mitochondria were destroyed in the hypoxic group, but were repaired in the crocin groups. The expressions of PGC-1α and SIRT1 were decreased in the hypoxic group, but were increased in the crocin group. All the effects improved by crocin were dose-dependent. Crocin attenuates acute hypobaric hypoxia-induced cognitive deficits in rats, accompanied by repairing the structures of hippocampal neurons and improving PGC-1α and SIRT1 levels. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Leukemia kidney infiltration can cause secondary polycythemia by activating hypoxia-inducible factor (HIF) pathway.

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    Osumi, Tomoo; Awazu, Midori; Fujimura, Eriko; Yamazaki, Fumito; Hashiguchi, Akinori; Shimada, Hiroyuki

    2013-06-01

    Secondary polycythemia with increased production of erythropoietin (EPO) is known to occur in kidney diseases such as hydronephrosis and cystic disease, but the mechanism remains unclear. We report an 18-year-old female with isolated renal relapse of acute lymphoblastic leukemia accompanied by polycythemia. At the relapse, she presented with bilateral nephromegaly, mild renal dysfunction, and erythrocytosis with increased serum EPO levels up to 52.1 mIU/mL (9.1-32.8). Renal biopsy demonstrated diffuse lymphoblastic infiltration. The expression of hypoxia-inducible factor (HIF)-1α, which is undetectable in normal kidney, was observed in the renal tubule epithelium compressed by lymphoblastic cells. These findings suggest that erythrocytosis was caused by renal ischemia due to leukemic infiltration. Polycythemia probably became apparent because of the lack of leukemic involvement of the bone marrow. With chemotherapy, the serum EPO level rapidly decreased to normal range accompanied by the normalization of kidney size and function. Renal leukemic infiltration may enhance EPO production, although not recognized in the majority of cases because of bone marrow involvement. Our case has clarified the mechanism of previously reported polycythemia associated with renal diseases as renal ischemia. Furthermore, we have added renal ischemia resulting from tumor infiltration to the list of causes of secondary polycythemia.

  20. Roles of hypoxia inducible factor-1α in the temporomandibular joint.

    Science.gov (United States)

    Mino-Oka, Akiko; Izawa, Takashi; Shinohara, Takehiro; Mori, Hiroki; Yasue, Akihiro; Tomita, Shuhei; Tanaka, Eiji

    2017-01-01

    Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative disease characterized by permanent cartilage loss. Articular cartilage is maintained in a low-oxygen environment. The chondrocyte response to hypoxic conditions involves expression of hypoxia inducible factor 1α (HIF-1α), which induces chondrocytes to increase expression of vascular endothelial growth factor (VEGF). Here, we investigated the role of HIF-1α in mechanical load effects on condylar cartilage and subchondral bone in heterozygous HIF-1α-deficient mice (HIF-1α+/-). Mechanical stress was applied to the TMJ of C57BL/6NCr wild-type (WT) and HIF-1α+/- mice with a sliding plate for 10 days. Histological analysis was performed by HE staining, Safranin-O/Fast green staining, and immunostaining specific for articular cartilage homeostasis. HIF-1α+/- mice had thinner cartilage and smaller areas of proteoglycan than WT controls, without and with mechanical stress. Mechanical stress resulted in prominent degenerative changes with increased expression of HIF-1α, VEGF, and the apoptosis factor cleaved Caspase-3 in condylar cartilage. Our results indicate that HIF-1α may be important for articular cartilage homeostasis and protective against articular cartilage degradation in the TMJ under mechanical stress condition, therefore HIF-1α could be an important new therapeutic target in TMJ-OA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Expression of hypoxia-inducible transcription factors in developing human and rat kidneys.

    Science.gov (United States)

    Bernhardt, W M; Schmitt, R; Rosenberger, C; Münchenhagen, P M; Gröne, H-J; Frei, U; Warnecke, C; Bachmann, S; Wiesener, M S; Willam, C; Eckardt, K-U

    2006-01-01

    Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.

  2. Hypoxia inducible factor: a potential prognostic biomarker in oral squamous cell carcinoma.

    Science.gov (United States)

    Qian, Jiang; Wenguang, Xu; Zhiyong, Wang; Yuntao, Zou; Wei, Han

    2016-08-01

    Oral squamous cell carcinoma (OSCC) is the most common oral cancer. Hypoxia inducible factor (HIF) is involved in many malignant tumors' growth and metastasis and upregulated by hypoxia, including oral cancer. Many studies have studied about the prognostic value of HIF expression in OSCC; however, they do not get the consistent results. Therefore, this study explored the correlation between the HIF expression and the prognosis of OSCC. It conducted a meta-analysis of relevant publications searched in the Web of Science, PubMed, and ISI Web of Knowledge databases. Totally, this study identified 12 relevant articles reporting a total of 1112 patients. This analysis revealed a significant association between increased risk of mortality (RR = 1.20; 95 % CI 0.74-1.95; I (2) 85.4 %) and overexpression of HIFs. Furthermore, different HIF isoforms were associated with overall survival [HIF-1α (RR = 1.18; 95 % CI 0.66-2.11; I (2) 87.2 %) and HIF-2α (RR = 1.40; 95 % CI 0.93-2.09; I(2) 0.0 %)]. These results show that overexpression of HIFs, regardless of whether the HIF-1α or HIF-2α isoforms are overexpressed is significantly associated with increased risk of mortality in OSCC patients. In this study, the funnel is symmetric, suggesting existed no publication bias.

  3. Crosstalk between nitric oxide and hypoxia-inducible factor signaling pathways: an update

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    Hendrickson MD

    2015-06-01

    Full Text Available Marina D Hendrickson, Robert O Poyton Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA Abstract: Hypoxia-inducible factor-1 (HIF-1 is responsible for cellular adaptations to hypoxia. While oxygen (O2 negatively regulates its stability, many other factors affect HIF-1 stability and activity, including nitric oxide (NO. NO derived from l-arginine and nitrite (NO2– could nitrosylate or nitrate HIF-1 and multiple proteins involved in HIF-1 regulation, and can allow HIF-1 to escape normoxic degradation. In turn, HIF-1 can increase NO production through multiple mechanisms, including increased inducible nitric oxide synthase (iNOS expression and subunit 4-2 of cytochrome c oxidase (COX4-2 expression. There is therefore a high degree of crosstalk between HIF-1 and NO signaling. As such, many cellular responses to NO are mediated by HIF-1, and vice versa. This includes, but is not limited to, angiogenesis, apoptosis, senescence, and metabolic changes. These pathways all have important functions in normal physiology and when altered can contribute or, in some cases, lead to pathogenesis. Keywords: HIF, nitric oxide, Cco/NO mitochondrial signaling, ROS/RNS, cancer

  4. Homodimerization of the PAS-B domains of hypoxia-inducible factors.

    Science.gov (United States)

    Zhu, Jing; Martinez-Yamout, Maria; Cardoso, Rosa; Yan, Jiangli; Love, Robert A; Grodsky, Neil; Brooun, Alexei; Dyson, H Jane

    2012-06-14

    The Per-Arnt-Sim (PAS) domains of hypoxia-inducible transcription factors (HIF) mediate heterodimer formation between the HIF-α forms that are induced in the event of cellular hypoxia and the constitutive HIF-β variants. Previous efforts toward structural characterization of the HIF-1α PAS domains were limited by protein stability. Using homology modeling based on the published crystal structure of the PAS-B domain of the homologous protein HIF-2α in complex with the partner HIF-β (also known as ARNT), we have identified a variant of HIF-1α with improved solubility, monodispersity, and stability. Purified solutions of the PAS-B domains of HIF-1α and HIF-2α differ in their propensity for homodimer formation. In an attempt to understand the structural basis for this difference, and to document the structural changes that accompany homodimer formation, we have undertaken a comparative NMR study of the PAS-B domains of HIF-1α and HIF-2α and mutants of HIF-1α that mimic the behavior of HIF-2α. The NMR spectra of all of these domains are very similar, consistent with the similarity of their amino acid sequences. However, the greater propensity of the HIF-1α PAS-B domain to form dimers as the concentration was increased allowed us to determine the site of homodimerization and pointed toward possible sequence changes in HIF-1α that might discourage the formation of homodimers.

  5. Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Mushfiquddin Khan

    2017-01-01

    Full Text Available Mild traumatic brain injury (TBI, also called concussion, initiates sequelae leading to motor deficits, cognitive impairments and subtly compromised neurobehaviors. While the acute phase of TBI is associated with neuroinflammation and nitroxidative burst, the chronic phase shows a lack of stimulation of the neurorepair process and regeneration. The deficiency of nitric oxide (NO, the consequent disturbed NO metabolome, and imbalanced mechanisms of S-nitrosylation are implicated in blocking the mechanisms of neurorepair processes and functional recovery in the both phases. Hypoxia inducible factor-1 alpha (HIF-1α, a master regulator of hypoxia/ischemia, stimulates the process of neurorepair and thus aids in functional recovery after brain trauma. The activity of HIF-1α is regulated by NO via the mechanism of S-nitrosylation of HIF-1α. S-nitrosylation is dynamically regulated by NO metabolites such as S-nitrosoglutathione (GSNO and peroxynitrite. GSNO stabilizes, and peroxynitrite destabilizes HIF-1α. Exogenously administered GSNO was found not only to stabilize HIF-1α and to induce HIF-1α-dependent genes but also to stimulate the regeneration process and to aid in functional recovery in TBI animals.

  6. hypoxia-inducible factors activate CD133 promoter through ETS family transcription factors.

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    Shunsuke Ohnishi

    Full Text Available CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1-P5 of CD133 in human embryonic kidney (HEK 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α. Deletion and mutation analysis identified one of the two E-twenty six (ETS binding sites (EBSs in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

  7. Anti-vascular agent Combretastatin A-4-P modulates Hypoxia Inducible Factor-1 and gene expression

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    Currie Margaret J

    2006-12-01

    Full Text Available Abstract Background A functional vascular network is essential for the survival, growth and spread of solid tumours, making blood vessels a key target for therapeutic strategies. Combretastatin A-4 phosphate (CA-4-P is a tubulin-depolymerising agent in Phase II clinical trials as a vascular disrupting agent. Not much is known of the molecular effect of CA-4-P under tumour conditions. The tumour microenvironment differs markedly from that in normal tissue, specifically with respect to oxygenation (hypoxia. Gene regulation under tumour conditions is governed by hypoxia inducible factor 1 (HIF-1, controlling angiogenic and metastatic pathways. Methods We investigated the effect of CA-4-P on factors of the upstream and downstream signalling pathway of HIF-1 in vitro. Results CA-4-P treatment under hypoxia tended to reduce HIF-1 accumulation in a concentration-dependent manner, an effect which was more prominent in endothelial cells than in cancer cell lines. Conversely, CA-4-P increased HIF-1 accumulation under aerobic conditions in vitro. At these concentrations of CA-4-P under aerobic conditions, nuclear factor κB was activated via the small GTPase RhoA, and expression of the HIF-1 downstream angiogenic effector gene, vascular endothelial growth factor (VEGF-A, was increased. Conclusion Our findings advance the understanding of signal transduction pathways involved in the actions of the anti-vascular agent CA-4-P.

  8. The importance of GLUT3 for de novo lipogenesis in hypoxia-induced lipid loading of human macrophages.

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    Lu Li

    Full Text Available Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.

  9. Effect of all-trans-retinoic acid on the development of chronic hypoxia-induced pulmonary hypertension.

    Science.gov (United States)

    Zhang, Erquan; Jiang, Baohua; Yokochi, Ayumu; Maruyama, Junko; Mitani, Yoshihide; Ma, Ning; Maruyama, Kazuo

    2010-08-01

    An earlier study showed that all-trans-retinoic acid (ATRA) prevents the development of monocrotalin-induced pulmonary hypertension (PH). The purpose of the present study was to determine the effect of ATRA on another model of chronic hypoxia-induced PH. Male Sprague-Dawley rats were given 30 mg/kg ATRA or vehicle only by gavage once daily for 14 days during hypobaric hypoxic exposure. Chronic hypoxic exposure induced PH, right ventricular hypertrophy (RVH), and hypertensive pulmonary vascular changes. Quantitative morphometry of the pulmonary arteries showed that ATRA treatment significantly reduced the percentage of muscularized arteries in peripheral pulmonary arteries only with an external diameter between 15 and 50 microm. ATRA treatment also significantly reduced the medial wall thickness in small muscular arteries only with an external diameter between 50 and 100 microm. Unfortunately, these reductions did not accompany the lowering of pulmonary artery pressure nor decrease in RVH. Chronic hypoxia-induced PH rats with ATRA had a loss in body weight. Chronic hypoxia increased the expression of endothelial nitric oxide synthase in the lung on western blotting and immunohistochemistry, in which ATRA treatment had no effect. The administration of ATRA might not have a therapeutic role in preventing the development of chronic hypoxia-induced PH, because of body weight loss and the subtle preventable effects of vascular changes.

  10. Salidroside attenuates chronic hypoxia-induced pulmonary hypertension via adenosine A2a receptor related mitochondria-dependent apoptosis pathway.

    Science.gov (United States)

    Huang, Xiaoying; Zou, Lizhen; Yu, Xiaoming; Chen, Mayun; Guo, Rui; Cai, Hui; Yao, Dan; Xu, Xiaomei; Chen, Yanfan; Ding, Cheng; Cai, Xueding; Wang, Liangxing

    2015-05-01

    Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Salidroside, an active ingredient isolated from Rhodiola rosea is proposed to exert protective effects against PAH. However, the function of salidroside in PAH has not been investigated systematically and the underlying mechanisms are not clear. To investigate the effects of salidroside on PAH, the mice in chronic hypoxia model of PAH were given by an increasing concentration of salidroside (0, 16 mg/kg, 32 mg/kg, and 64 mg/kg). After salidroside treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodeling were attenuated, suggesting a protective role played by salidroside in PAH. To explore the potential mechanisms, the apoptosis of PASMCs after salidroside treatment under hypoxia conditions were determined in vivo and in vitro, and also the mitochondria-dependent apoptosis factors, Bax, Bcl-2, cytochrome C, and caspase 9 were examined. The results revealed that salidroside reversed hypoxia-induced cell apoptosis resistance at least partially via a mitochondria-dependent pathway. In addition, salidroside upregulated the expression of adenosine A2a receptor (A2aR) in lung tissues of mice and in PASMCs in vitro after hypoxia exposure. Combined the evidence above, we conclude that salidroside can attenuate chronic hypoxia-induced PAH by promoting PASMCs apoptosis via an A2aR related mitochondria dependent pathway. Copyright © 2015. Published by Elsevier Ltd.

  11. Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qiang [Department of Neurology, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Department of Neurology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Zhang, Ting [Department of Neurology, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Wang, Jixian [Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030 (China); Zhang, Zhijun [Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030 (China); Zhai, Yu [Department of Neurology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Yang, Guo-Yuan, E-mail: gyyang0626@gmail.com [Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030 (China); Sun, Xiaojiang, E-mail: sunxj19@gmail.com [Department of Neurology, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China)

    2014-02-07

    Highlights: • Rapamycin enhances mitophagy via increasing p62 translocation to the mitochondria. • Rapamycin attenuates brain ischemic damage and improves mitochondrial function. • The protection of rapamycin to mitochondrial is linked to enhanced mitophagy. - Abstract: Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model. However, the involvement of mitophagy in this process and its contribution to the protection of mitochondrial function remains unknown. The present study explored the characteristics of mitophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function. Male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO). Neurological deficits scores; infarct volumes; mitophagy morphology; and the levels of malondialdehyde (MDA), adenosine triphosphate (ATP) and mitochondrial membrane potentials (Δψm) were examined. The expression of LC3, Beclin-1 and p62 in the mitochondrial fraction combined with transmission electronic microscopy were used to explore mitophagic activity after ischemia. We also blocked autophagosome formation using 3-methyladenine (3-MA) to check the linkage between the mitochondrial protective effect of rapamycin and enhanced mitophagy. We observed that rapamycin significantly enhanced mitophagy, as evidenced by the increase in LC3-II and Beclin-1 expression in the mitochondria and p62 translocation to the mitochondria. Rapamycin reduced infarct volume, improved neurological outcomes and inhibited mitochondrial dysfunction compared with the control animals (p < 0.05). However, these protective effects were reversed by 3-methyladenine treatment after rapamycin. The present study indicates that rapamycin treatment attenuates mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced mitophagy.

  12. Rapamycin prevents drug seeking via disrupting reconsolidation of reward memory in rats.

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    Lin, Jue; Liu, Lingqi; Wen, Quan; Zheng, Chunming; Gao, Yang; Peng, Shuxian; Tan, Yalun; Li, Yanqin

    2014-01-01

    The maladaptive drug memory developed between the drug-rewarding effect and environmental cues contributes to difficulty in preventing drug relapse. Established reward memories can be disrupted by pharmacologic interventions following their reactivation. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, has been proved to be involved in various memory consolidation. However, it is less well characterized in drug memory reconsolidation. Using a conditioned place preference (CPP) procedure, we examined the effects of systemically administered rapamycin on reconsolidation of drug memory in rats. We found that systemically administered rapamycin (0.1 or 10 mg/kg, i.p.) after re-exposure to drug-paired environment, dose dependently decreased the expression of CPP 1 d later, and the effect lasted for up to 14 d and could not be reversed by a priming injection of morphine. The effect of rapamycin on morphine-associated memory was specific to drug-paired context, and rapamycin had no effect on subsequent CPP expression when rats were exposed to saline-paired context or homecage. These results indicated that systemic administration of rapamycin after memory reactivation can persistently inhibit the drug seeking behaviour via disruption of morphine memory reconsolidation in rats. Additionally, the effect of rapamycin on memory reconsolidation was reproduced in cocaine CPP and alcohol CPP. Furthermore, rapamycin did not induce conditioned place aversion and had no effect on locomotor activity and anxiety behaviour. These findings suggest that rapamycin could erase the acquired drug CPP in rats, and that mTOR activity plays an important role in drug reconsolidation and is required for drug relapse.

  13. Rapamycin promotes osteogenesis under inflammatory conditions.

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    Li, Xing; Chang, Bei; Wang, Banchao; Bu, Wenhuan; Zhao, Liang; Liu, Jie; Meng, Lin; Wang, Lu; Xin, Ying; Wang, Dandan; Tang, Qi; Zheng, Changyu; Sun, Hongchen

    2017-12-01

    Chronic periodontitis, a common oral disease, usually results in irreversible bone resorption. Bone regeneration is a complex process between bone‑forming activity of osteoblasts and bone‑resorbing activity of osteoclasts, and still remains a challenge for physicians clinically. A previous study demonstrated that the mechanistic target of rapamycin signaling pathway is involved in osteogenic differentiation of mesenchymal stromal cells. Herein, whether rapamycin could be used to induce osteogenic differentiation of primary bone marrow‑derived mesenchymal stem cells (BMSCs) in vitro and promote new bone formation in vivo were evaluated. The results demonstrated that rapamycin alone was not enough to fully induce osteoblast differentiation in vitro and enhanced bone regeneration in vivo. Interestingly, rapamycin in rapamycin plus lipopolysaccharide (LPS)‑treated BMSCs significantly increased the gene expression levels of Sp7 transcription factor, runt related transcription factor 2, alkaline phosphatase (ALP) and collagen I (Col I), ALP activity, and calcium nodule at different time points in vitro, indicating that osteoblast differentiation occurs by rapamycin when BMSCs are exposed to LPS simultaneously. It was also demonstrated that rapamycin in rapamycin plus LPS‑treated rats promoted bone regeneration in vivo. These results suggest that rapamycin may influence osteoblast differentiation and new bone formation after LPS induces an inflammatory environment. Rapamycin may be used to treat periodontitis associated with bone loss in future clinical practice.

  14. The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression.

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    Park, Mi Sun; Kim, Boh-Ram; Dong, Seung Myung; Lee, Seung-Hoon; Kim, Dae-Yong; Rho, Seung Bae

    2014-07-15

    Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation in vitro. Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.

  15. [Effects of RNAi on hypoxia inducible factor-1alpha activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rat].

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    Zhang, Wei; Cao, Yue; Zhang, Yu; Ma, Qi-Sheng; Ma, Lan; Ge, Ri-Li

    2006-02-25

    Pulmonary vascular remodeling is one of the major characteristics of hypoxia-induced pulmonary hypertension, mainly represented by over-proliferation of pulmonary artery smooth muscle cells (PASMCs). Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor which is produced by the cells exposed to hypoxia. HIF-1alpha up-regulates the expression of many hypoxia response genes (HRGs) for the body to adapt to hypoxia and maintain homeostasis. The expression of HIF-1alpha in the PASMCs is remarkably elevated under hypoxic condition and it stimulates the proliferation of PASMCs. In this experiment, we used gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1alpha mRNA. They were separately subcloned into the plasmid of pGenesil-1 containing U6 promoter. The pGenesil-1 vector of the RNA interference eukaryotic expression vector specific to HIF-1alpha gene was constructed. DNA sequencing of the plasmid verified the successful construction of the HIF-1alpha RNAi. We isolated and cultured the PASMCs of rat. The pGenesil-1 vector was transferred into the PASMCs with METAFECTENE in vitro. The positive cell clones transfected with pGenesil-1 were obtained after being screened with 400 mug/ml G418. These PASMCs were cultured in normoxia and hypoxia. After 48 h, the effects of RNAi on the expression of HIF-1alpha mRNA were detected by RT-PCR. The cellular growth activities were assayed by MTT colorimetry and flow cytometry in vitro. The results showed that for the PASMCs cultured in hypoxia for 48 h, the cell proliferation of blank group and control group were remarkably increased and the HIF-1alpha mRNA expressions were up-regulated, while the cell proliferation of the treatment groups did not increase and the HIF-1alpha mRNA expressions were not up-regulated. In conclusion, we successfully constructed the recombinant plasmid of RNAi and transfected them into the PASMCs in vitro. The RNAi inhibited the expression of HIF-1alpha m

  16. Rapamycin Ameliorates Proteinuria and Restores Nephrin and Podocin Expression in Experimental Membranous Nephropathy

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    Stavros Stratakis

    2013-01-01

    Full Text Available Objective. Recent studies have shown a beneficial effect of rapamycin in passive and active Heymann Nephritis (HN. However, the mechanisms underlying this beneficial effect have not been elucidated. Methods. Passive Heymann Nephritis (PHN was induced by a single intravenous infusion of anti-Fx1 in 12 Sprague-Dawley male rats. One week later, six of these rats were commenced on daily treatment with subcutaneous rapamycin 0.5 mgr/kg (PHN-Rapa. The remaining six rats were used as the proteinuric control group (PHN while six more rats without PHN were given the rapamycin solvent and served as the healthy control group (HC. All rats were sacrificed at the end of the 7th week. Results. Rapamycin significantly reduced proteinuria during the autologous phase of PHN. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression. Conclusions. Rapamycin monotherapy significantly improves proteinuria and histological lesions in experimental membranous nephropathy. This beneficial effect may be mediated by inhibition of the alloimmune response during the autologous phase of PHN and by restoration of the normal expression of the podocyte proteins nephrin and podocin.

  17. Suppression of hypoxia-inducible factor-1alpha and its downstream genes reduces acute hyperglycemia-enhanced hemorrhagic transformation in a rat model of cerebral ischemia.

    Science.gov (United States)

    Chen, Chunhua; Ostrowski, Robert P; Zhou, Changman; Tang, Jiping; Zhang, John H

    2010-07-01

    We evaluated a role of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream genes in acute hyperglycemia-induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats weighing 280-300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF-1alpha inhibitors, 2-methoxyestradiol (2ME2) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF-1alpha inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF-1alpha was increased in ischemic brain tissues after MCAO and reduced by HIF-1alpha inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase-2 and -9 (MMP-2/MMP-9) in the ipsilateral hemisphere. Both 2ME2 and YC-1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF-1alpha and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia.

  18. Modeling the dynamics of hypoxia inducible factor-1α (HIF-1α) within single cells and 3D cell culture systems.

    Science.gov (United States)

    Leedale, Joseph; Herrmann, Anne; Bagnall, James; Fercher, Andreas; Papkovsky, Dmitri; Sée, Violaine; Bearon, Rachel N

    2014-12-01

    HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that mediates the intracellular response to hypoxia in human cells. There is increasing evidence that cell signaling pathways encode temporal information, and thus cell fate may be determined by the dynamics of protein levels. We have developed a mathematical model to describe the transient dynamics of the HIF-1α protein measured in single cells subjected to hypoxic shock. The essential characteristics of these data are modeled with a system of differential equations describing the feedback inhibition between HIF-1α and prolyl hydroxylases (PHD) oxygen sensors. Heterogeneity in the single-cell data is accounted through parameter variation in the model. We previously identified the PHD2 isoform as the main PHD sensor responsible for controlling the HIF-1α transient response, and make here testable predictions regarding HIF-1α dynamics subject to repetitive hypoxic pulses. The model is further developed to describe the dynamics of HIF-1α in cells cultured as 3D spheroids, with oxygen dynamics parameterized using experimental measurements of oxygen within spheroids. We show that the dynamics of HIF-1α and transcriptional targets of HIF-1α display a non-monotone response to the oxygen dynamics. Specifically we demonstrate that the dynamic transient behavior of HIF-1α results in differential dynamics in transcriptional targets. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. The PPARbeta/delta agonist GW0742 relaxes pulmonary vessels and limits right heart hypertrophy in rats with hypoxia-induced pulmonary hypertension.

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    Louise S Harrington

    2010-03-01

    Full Text Available Pulmonary vascular diseases are increasingly recognised as important clinical conditions. Pulmonary hypertension associated with a range of aetiologies is difficult to treat and associated with progressive morbidity and mortality. Current therapies for pulmonary hypertension include phosphodiesterase type 5 inhibitors, endothelin receptor antagonists, or prostacyclin mimetics. However, none of these provide a cure and the clinical benefits of these drugs individually decline over time. There is, therefore, an urgent need to identify new treatment strategies for pulmonary hypertension.Here we show that the PPARbeta/delta agonist GW0742 induces vasorelaxation in systemic and pulmonary vessels. Using tissue from genetically modified mice, we show that the dilator effects of GW0742 are independent of the target receptor PPARbeta/delta or cell surface prostacyclin (IP receptors. In aortic tissue, vascular relaxant effects of GW0742 were not associated with increases in cGMP, cAMP or hyperpolarisation, but were attributed to inhibition of RhoA activity. In a rat model of hypoxia-induced pulmonary hypertension, daily oral dosing of animals with GW0742 (30 mg/kg for 3 weeks significantly reduced the associated right heart hypertrophy and right ventricular systolic pressure. GW0742 had no effect on vascular remodelling induced by hypoxia in this model.These observations are the first to show a therapeutic benefit of 'PPARbeta/delta' agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.

  20. Hypoxia-Inducible Factor-1α: A Potential Factor for the Enhancement of Osseointegration between Dental Implants and Tissue-Engineered Bone

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    Duohong Zou

    2011-07-01

    Full Text Available Introduction: Tissue-engineered bones are widely utilized to protect healthy tissue, reduce pain, and increase the success rate of dental implants. one of the most challenging obstacles lies in obtaining effective os-seointegration between dental implants and tissue-engineered structures. Deficiencies in vascularization, osteogenic factors, oxygen, and other nutrients inside the tissue-engineered bone during the early stages following implantation all inhibit effective osseointe-gration. Oxygen is required for aerobic metabolism in bone and blood vessel tissues, but oxygen levels inside tissue-engineered bone are not suf-ficient for cell proliferation. HIF-1α is a pivotal regulator of hypoxic and ischemic vascular responses, driving transcriptional activation of hundreds of genes involved in vascular reactivity, angiogenesis, arteriogenesis, and osteogenesis.The hypothesis: Hypoxia-Inducible Factor-1α seems a potential factor for the enhancement of osseointegration between dental implants and tissue-engineered bone.Evaluation of the hypothesis: Enhancement of HIF-1α protein expression is recognized as the most promising approach for angiogenesis, because it can induce multiple angiogenic targets in a coordinated manner. Therefore, it will be a novel potential therapeutic methods targeting HIF-1α expression to enhance osseointegration be-tween dental implants and tissue-engineered bone.

  1. Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin.

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    Butler, Corwin R; Boychuk, Jeffery A; Smith, Bret N

    2017-01-01

    Following traumatic brain injury (TBI), treatment with rapamycin suppresses mammalian (mechanistic) target of rapamycin (mTOR) activity and specific components of hippocampal synaptic reorganization associated with altered cortical excitability and seizure susceptibility. Reemergence of seizures after cessation of rapamycin treatment suggests, however, an incomplete suppression of epileptogenesis. Hilar inhibitory interneurons regulate dentate granule cell (DGC) activity, and de novo synaptic input from both DGCs and CA3 pyramidal cells after TBI increases their excitability but effects of rapamycin treatment on the injury-induced plasticity of interneurons is only partially described. Using transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in the somatostatinergic subset of hilar inhibitory interneurons, we tested the effect of daily systemic rapamycin treatment (3 mg/kg) on the excitability of hilar inhibitory interneurons after controlled cortical impact (CCI)-induced focal brain injury. Rapamycin treatment reduced, but did not normalize, the injury-induced increase in excitability of surviving eGFP+ hilar interneurons. The injury-induced increase in response to selective glutamate photostimulation of DGCs was reduced to normal levels after mTOR inhibition, but the postinjury increase in synaptic excitation arising from CA3 pyramidal cell activity was unaffected by rapamycin treatment. The incomplete suppression of synaptic reorganization in inhibitory circuits after brain injury could contribute to hippocampal hyperexcitability and the eventual reemergence of the epileptogenic process upon cessation of mTOR inhibition. Further, the cell-selective effect of mTOR inhibition on synaptic reorganization after CCI suggests possible mechanisms by which rapamycin treatment modifies epileptogenesis in some models but not others.

  2. Chronic intermittent hypoxia induces cardiac inflammation and dysfunction in a rat obstructive sleep apnea model.

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    Wei, Qin; Bian, Yeping; Yu, Fuchao; Zhang, Qiang; Zhang, Guanghao; Li, Yang; Song, Songsong; Ren, Xiaomei; Tong, Jiayi

    2016-11-01

    Chronic intermittent hypoxia is considered to play an important role in cardiovascular pathogenesis during the development of obstructive sleep apnea (OSA). We used a well-described OSA rat model induced with simultaneous intermittent hypoxia. Male Sprague Dawley rats were individually placed into plexiglass chambers with air pressure and components were electronically controlled. The rats were exposed to intermittent hypoxia 8 hours daily for 5 weeks. The changes of cardiac structure and function were examined by ultrasound. The cardiac pathology, apoptosis, and fibrosis were analyzed by H&E staining, TUNNEL assay, and picosirius staining, respectively. The expression of inflammation and fibrosis marker genes was analyzed by quantitative real-time PCR and Western blot. Chronic intermittent hypoxia/low pressure resulted in significant increase of left ventricular internal diameters (LVIDs), end-systolic volume (ESV), end-diastolic volume (EDV), and blood lactate level and marked reduction in ejection fraction and fractional shortening. Chronic intermittent hypoxia increased TUNNEL-positive myocytes, disrupted normal arrangement of cardiac fibers, and increased Sirius stained collagen fibers. The expression levels of hypoxia induced factor (HIF)-1α, NF-kB, IL-6, and matrix metallopeptidase 2 (MMP-2) were significantly increased in the heart of rats exposed to chronic intermittent hypoxia. In conclusion, the left ventricular function was adversely affected by chronic intermittent hypoxia, which is associated with increased expression of HIF-1α and NF-kB signaling molecules and development of cardiac inflammation, apoptosis and fibrosis. © 2016 by the Journal of Biomedical Research. All rights reserved.

  3. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism.

    Science.gov (United States)

    Elks, Philip M; Brizee, Sabrina; van der Vaart, Michiel; Walmsley, Sarah R; van Eeden, Fredericus J; Renshaw, Stephen A; Meijer, Annemarie H

    2013-01-01

    Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic

  4. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism.

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    Philip M Elks

    Full Text Available Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb, becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for

  5. Identification of hypoxia-induced genes in human SGBS adipocytes by microarray analysis.

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    Kathrin Geiger

    Full Text Available Hypoxia in adipose tissue is suggested to be involved in the development of a chronic mild inflammation, which in obesity can further lead to insulin resistance. The effect of hypoxia on gene expression in adipocytes appears to play a central role in this inflammatory response observed in obesity. However, the global impact of hypoxia on transcriptional changes in human adipocytes is unclear. Therefore, we compared gene expression profiles of human Simpson-Golabi-Behmel syndrome (SGBS adipocytes under normoxic or hypoxic conditions to detect hypoxia-responsive genes in adipocytes by using whole human genome microarrays. Microarray analysis showed more than 500 significantly differentially regulated mRNAs after incubation of the cells under low oxygen levels. To gain further insight into the biological processes, hypoxia-regulated genes after 16 hours of hypoxia were classified according to their function. We identified an enrichment of genes involved in important biological processes such as glycolysis, response to hypoxia, regulation of cellular component movement, response to nutrient levels, regulation of cell migration, and transcription regulator activity. Real-time PCR confirmed eight genes to be consistently upregulated in response to 3, 6 and 16 hours of hypoxia. For adipocytes the hypoxia-induced regulation of these genes is shown here for the first time. Moreover in six of these eight genes we identified HIF response elements in the proximal promoters, specific for the HIF transcription factor family members HIF1A and HIF2A. In the present study, we demonstrated that hypoxia has an extensive effect on gene expression of SGBS adipocytes. In addition, the identified hypoxia-regulated genes are likely involved in the regulation of obesity, the incidence of type 2 diabetes, and the metabolic syndrome.

  6. mRNA cycles through hypoxia-induced stress granules in live Drosophila embryonic muscles.

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    van der Laan, Annelies M A; van Gemert, Alice M C; Dirks, Roeland W; Noordermeer, Jasprina N; Fradkin, Lee G; Tanke, Hans J; Jost, Carolina R

    2012-01-01

    In some myopathies, hypoxia can be the result of pathologic effects like muscle necrosis and abnormal blood flow. At the molecular level, the consequence of hypoxic conditions is not yet fully understood. Under stress conditions, many housekeeping gene mRNAs are translationally silenced, while translation of other mRNAs increases. Alterations to the pool of mRNAs available for translation lead to the formation of so-called stress granules containing both mRNAs and proteins. Stress granule formation and dynamics have been investigated using cells in culture, but have not yet been examined in vivo. In Drosophila embryonic muscles, we found that hypoxia induces the formation of sarcoplasmic granules containing the established stress granule markers RIN and dFMR1. Upon restoration of normoxia, the observed granules were decreased in size, indicating that their formation might be reversible. Employing photobleaching approaches, we found that a cytoplasmic reporter mRNA rapidly shuttles in and out of the granules. Hence, stress granules are highly dynamic complexes and not simple temporary storage sites. Although mRNA rapidly cycles through the granules, its movement throughout the muscle is, remarkably, spatially restricted by the presence of yet undefined myofiber domains. Our results suggest that in hypoxic muscles mRNA remains highly mobile; however, its movement throughout the muscle is restricted by certain boundaries. The development of this Drosophila hypoxia model makes it possible to study the formation and dynamics of stress granules and their associated mRNAs and proteins in a living organism.

  7. Integrative genomics reveals hypoxia inducible genes that are associated with a poor prognosis in neuroblastoma patients

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    Kao, Clara; Hernandez, Kyle M.; DeWane, Gillian; Salwen, Helen R.; Chlenski, Alexandre; Dobratic, Marija; Mariani, Christopher J.; Godley, Lucy A.; Prabhakar, Nanduri; White, Kevin; Stranger, Barbara E.; Cohn, Susan L.

    2016-01-01

    Neuroblastoma is notable for its broad spectrum of clinical behavior ranging from spontaneous regression to rapidly progressive disease. Hypoxia is well known to confer a more aggressive phenotype in neuroblastoma. We analyzed transcriptome data from diagnostic neuroblastoma tumors and hypoxic neuroblastoma cell lines to identify genes whose expression levels correlate with poor patient outcome and are involved in the hypoxia response. By integrating a diverse set of transcriptome datasets, including those from neuroblastoma patients and neuroblastoma derived cell lines, we identified nine genes (SLCO4A1, ENO1, HK2, PGK1, MTFP1, HILPDA, VKORC1, TPI1, and HIST1H1C) that are up-regulated in hypoxia and whose expression levels are correlated with poor patient outcome in three independent neuroblastoma cohorts. Analysis of 5-hydroxymethylcytosine and ENCODE data indicate that at least five of these nine genes have an increase in 5-hydroxymethylcytosine and a more open chromatin structure in hypoxia versus normoxia and are putative targets of hypoxia inducible factor (HIF) as they contain HIF binding sites in their regulatory regions. Four of these genes are key components of the glycolytic pathway and another three are directly involved in cellular metabolism. We experimentally validated our computational findings demonstrating that seven of the nine genes are significantly up-regulated in response to hypoxia in the four neuroblastoma cell lines tested. This compact and robustly validated group of genes, is associated with the hypoxia response in aggressive neuroblastoma and may represent a novel target for biomarker and therapeutic development. PMID:27765905

  8. Expression of hypoxia-inducible factor-1α and erythropoietin at corneal neovascularization in rats

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    Ji-Min Wang

    2014-12-01

    Full Text Available AIM: To describe the expression of hypoxia-inducible factor-1α(HIF-1αand erythropoietin(EPOin rats' corneal and evaluate its potential effect on corneal neovascularization(CNVgrowth. METHODS: The young SD rats(3mowas chosen and randomly divided into 2 groups, which were experimental group and normal control group. CNV model was established by alkali burn, and the length and area of CNV was observed everyday after operation by slit lamp. After that, the expression of HIF-1α and EPO was measured by SABC and RT-PCR methods at 1, 3, 5, 7, and 14d after alkali burn. The data was analyzed by SPSS 20.0. RESULTS: The area of CNV was increasing at 1, 3, 5, 7, and 14d after alkali burn, and the peak point appear at 7d. The growth speed was decreased after 14d. SABC method told us that no HIF-1α and very tiny amount EPO was detected at normal rats' corneal. The expression of the two factors increased at 1d after alkali burn in corneal epithelium and endoderm. The results of RT-PCR showed that a few amounts of HIF-1α and EPO mRNA were detected at normal group. The expression of the two factors was increased at 3d after alkali burn, and the peak value was found at 7d, however, it was decreased at 14d. Statistical difference was found at different time(PCONCLUSION: HIF-1α and EPO is closely related to CNV.

  9. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

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    Ziegler Andreas

    2005-09-01

    Full Text Available Abstract Background Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling. Methods and Results Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO2 = 0.1 using nylon filters (1176 spots. Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-β, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media. Conclusion Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.

  10. Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice

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    Izziki Mohamed

    2009-01-01

    Full Text Available Abstract Background Inflammation may contribute to the pathogenesis of various forms of pulmonary hypertension (PH. Recent studies in patients with idiopathic PH or PH associated with underlying diseases suggest a role for interleukin-6 (IL-6. Methods To determine whether endogenous IL-6 contributes to mediate hypoxic PH and lung inflammation, we studied IL-6-deficient (IL-6-/- and wild-type (IL-6+/+ mice exposed to hypoxia for 2 weeks. Results Right ventricular systolic pressure, right ventricle hypertrophy, and the number and media thickness of muscular pulmonary vessels were decreased in IL-6-/- mice compared to wild-type controls after 2 weeks' hypoxia, although the pressure response to acute hypoxia was similar in IL-6+/+ and IL-6-/- mice. Hypoxia exposure of IL-6+/+ mice led to marked increases in IL-6 mRNA and protein levels within the first week, with positive IL-6 immunostaining in the pulmonary vessel walls. Lung IL-6 receptor and gp 130 (the IL-6 signal transducer mRNA levels increased after 1 and 2 weeks' hypoxia. In vitro studies of cultured human pulmonary-artery smooth-muscle-cells (PA-SMCs and microvascular endothelial cells revealed prominent synthesis of IL-6 by PA-SMCs, with further stimulation by hypoxia. IL-6 also markedly stimulated PA-SMC migration without affecting proliferation. Hypoxic IL-6-/- mice showed less inflammatory cell recruitment in the lungs, compared to hypoxic wild-type mice, as assessed by lung protein levels and immunostaining for the specific macrophage marker F4/80, with no difference in lung expression of adhesion molecules or cytokines. Conclusion These data suggest that IL-6 may be actively involved in hypoxia-induced lung inflammation and pulmonary vascular remodeling in mice.

  11. Hypoxia Inducible Factor (HIF transcription factor family expansion, diversification, divergence and selection in eukaryotes.

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    Allie M Graham

    Full Text Available Hypoxia inducible factor (HIF transcription factors are crucial for regulating a variety of cellular activities in response to oxygen stress (hypoxia. In this study, we determine the evolutionary history of HIF genes and their associated transactivation domains, as well as perform selection and functional divergence analyses across their four characteristic domains. Here we show that the HIF genes are restricted to metazoans: At least one HIF-α homolog is found within the genomes of non-bilaterians and bilaterian invertebrates, while most vertebrate genomes contain between two and six HIF-α genes. We also find widespread purifying selection across all four characteristic domain types, bHLH, PAS, NTAD, CTAD, in HIF-α genes, and evidence for Type I functional divergence between HIF-1α, HIF-2α /EPAS, and invertebrate HIF genes. Overall, we describe the evolutionary histories of the HIF transcription factor gene family and its associated transactivation domains in eukaryotes. We show that the NTAD and CTAD domains appear de novo, without any appearance outside of the HIF-α subunits. Although they both appear in invertebrates as well as vertebrate HIF- α sequences, there seems to have been a substantial loss across invertebrates or were convergently acquired in these few lineages. We reaffirm that HIF-1α is phylogenetically conserved among most metazoans, whereas HIF-2α appeared later. Overall, our findings can be attributed to the substantial integration of this transcription factor family into the critical tasks associated with maintenance of oxygen homeostasis and vascularization, particularly in the vertebrate lineage.

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

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    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.

  13. Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye.

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    Ji Hwan Min

    Full Text Available By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO mice and a dry eye (DE mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs.C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45+ cells in LGs was assessed by immunohistochemical (IHC staining and flow cytometry for each condition.DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45+ cell infiltration in LGs. Likewise, CD45+ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice.Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.

  14. Effects of YC-1 on hypoxia-inducible factor 1 alpha in hypoxic human bladder transitional carcinoma cell line T24 cells.

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    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.

  15. The volatile anesthetic isoflurane differentially suppresses the induction of erythropoietin synthesis elicited by acute anemia and systemic hypoxemia in mice in an hypoxia-inducible factor-2-dependent manner.

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    Kai, Shinichi; Tanaka, Tomoharu; Matsuyama, Tomonori; Suzuki, Kengo; Hirota, Kiichi

    2014-06-05

    Erythropoietin (EPO) is a glycoprotein hormone essential for the regulation of erythroid homeostasis. Although EPO production is prominent in the kidney and liver, its production in the central nervous system has also been detected. Tissue hypoxia due to systemic or local hypoxemia and acute anemia due to blood loss occurs frequently during various clinical settings, leading to a high possibility of elevated plasma EPO levels. However, it is largely unknown whether volatile anesthetic agents affect EPO production elicited by acute hypoxia in vivo. Male C57BL/6N CrSlc mice were exposed to a hypoxic insult as a result of bleeding-related anemia or hypoxemia while they were under anesthetized using various concentrations of isoflurane. EPO protein concentrations were assessed by enzyme-linked immunosorbent assay and mRNA levels were measured by quantitative real-time reverse transcriptase-polymerase chain reaction. Plasma EPO concentration was induced as early as 3h following acute anemic and hypoxemic hypoxia and suppressed by clinically relevant doses of isoflurane in a dose-dependent manner. Anemic hypoxia induced EPO mRNA and protein synthesis in the kidney. In the kidney, isoflurane inhibited EPO induction caused by anemia but not that caused by hypoxemia. On the other hand, in the brain hypoxemia-induced EPO production was suppressed by isoflurane. Finally, qRT-PCR studies demonstrate that isoflurane differentially inhibit HIF-1α and HIF-2α mRNA expression in brain and kidney, indicating the involvement of HIF-2 in the hypoxia-induced EPO expression and inhibition of the induction by isoflurane. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Rapamycin reversal of VEGF-C-driven lymphatic anomalies in the respiratory tract.

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    Baluk, Peter; Yao, Li-Chin; Flores, Julio C; Choi, Dongwon; Hong, Young-Kwon; McDonald, Donald M

    2017-08-17

    Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, β-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C-driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

  17. ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.

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    Melissa M Keenan

    2015-10-01

    Full Text Available In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1 or ATP citrate lyase (ACLY protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

  18. Proteomic analysis reveals that proteasome subunit beta 6 is involved in hypoxia-induced pulmonary vascular remodeling in rats.

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    Jian Wang

    Full Text Available Chronic hypoxia (CH is known to be one of the major causes of pulmonary hypertension (PH, which is characterized by sustained elevation of pulmonary vascular resistance resulting from vascular remodeling. In this study, we investigated whether the ubiquitin proteasome system (UPS was involved in the mechanism of hypoxia-induced pulmonary vascular remodeling. We isolated the distal pulmonary artery (PA from a previously defined chronic hypoxic pulmonary hypertension (CHPH rat model, performed proteomic analyses in search of differentially expressed proteins belonging to the UPS, and subsequently identified their roles in arterial remodeling.Twenty-two proteins were differently expressed between the CH and normoxic group. Among them, the expression of proteasome subunit beta (PSMB 1 and PSMB6 increased after CH exposure. Given that PSMB1 is a well-known structural subunit and PSMB6 is a functional subunit, we sought to assess whether PSMB6 could be related to the multiple functional changes during the CHPH process. We confirmed the proteomic results by real-time PCR and Western blot. With the increase in quantity of the active subunit, proteasome activity in both cultured pulmonary artery smooth muscle cells (PASMCs and isolated PA from the hypoxic group increased. An MTT assay revealed that the proteasome inhibitor MG132 was able to attenuate the hypoxia-induced proliferation of PASMC in a dose-dependent manner. Knockdown of PSMB6 using siRNA also prevented hypoxia-induced proliferation.The present study revealed the association between increased PSMB6 and CHPH. CH up-regulated proteasome activity and the proliferation of PASMCs, which may have been related to increased PSMB6 expression and the subsequently enhanced functional catalytic sites of the proteasome. These results suggested an essential role of the proteasome during CHPH development, a novel finding requiring further study.

  19. Differential Effects of Rapamycin and Dexamethasone in Mouse Models of Established Allergic Asthma

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    Mushaben, Elizabeth M.; Brandt, Eric B.; Hershey, Gurjit K. Khurana; Le Cras, Timothy D.

    2013-01-01

    The mammalian target of rapamycin (mTOR) plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM)-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections) and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4–6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR), inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4+ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4+ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established. PMID:23349887

  20. Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma.

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    Tim F Cloughesy

    2008-01-01

    Full Text Available There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR, we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation varied substantially. Tumor cell proliferation (measured by Ki-67 staining was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test.Rapamycin

  1. A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

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    Wen Bixiu; Burgman, Paul; Zanzonico, Pat; O' Donoghue, Joseph; Li, Gloria C.; Ling, C. Clifton [Memorial Sloan-Kettering Cancer Center, Department of Medical Physics, New York (United States); Cai Shangde; Finn, Ron [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Serganova, Inna [Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States); Blasberg, Ronald; Gelovani, Juri [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States)

    2004-11-01

    Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the {sup 124}I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil ({sup 124}I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped {sup 124}I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of {sup 124}I-FIAU was also compared with that of an exogenous hypoxic cell marker, {sup 18}F-fluoromisonidazole (FMISO). Our results showed that {sup 124}I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of {sup 124}I-FIAU and {sup 18}F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between {sup 124}I-FIAU and {sup 18}F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future

  2. Retraction: Genistein protects genioglossus myocyte against hypoxia-induced injury through PI3K-Akt and ERK MAPK pathways.

    Science.gov (United States)

    2012-05-01

    RETRACTION: The following article from Journal of Cellular Biochemistry, Genistein protects genioglossus myocyte against hypoxia-induced injury through PI3K-Akt and ERK MAPK pathways by Wanghui Ding and Yuehua Liu, posted online on May 19, 2011 in Wiley Online Library (onlinelibrary.wiley.com), has been retracted by agreement between the authors, the journal Editor in Chief, Dr. Gary S. Stein and Wiley-Liss, Inc. The retraction has been made as authorization to publish was not granted by one of the funding bodies. Copyright © 2012 Wiley Periodicals, Inc.

  3. [Effects of mTOR Inhibitor Rapamycin on Burkitt's Lymphoma Cells].

    Science.gov (United States)

    Zhou, Lun-Huan; Zhu, Xiong-Peng; Xiao, Hui-Fang; Xin, Peng-Liang; Li, Chun-Tuan

    2017-10-01

    To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin. Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G1/G0 phase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G2/M phase, the decreased population was accompanied by the increase of G1/G0 phase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6. The rapamycin can effectively inhibit cell proliferation

  4. Mammalian-target of rapamycin inhibition with temsirolimus in myelodysplastic syndromes (MDS) patients is associated with considerable toxicity: results of the temsirolimus pilot trial by the German MDS Study Group (D-MDS).

    Science.gov (United States)

    Wermke, Martin; Schuster, Claudia; Nolte, Florian; Al-Ali, Haifa-Kathrin; Kiewe, Philipp; Schönefeldt, Claudia; Jakob, Christiane; von Bonin, Malte; Hentschel, Leopold; Klut, Ina-Maria; Ehninger, Gerhard; Bornhäuser, Martin; Baretton, Gustavo; Germing, Ulrich; Herbst, Regina; Haase, Detelef; Hofmann, Wolf K; Platzbecker, Uwe

    2016-12-01

    The mammalian-target of rapamycin (also termed mechanistic target of rapamycin, mTOR) pathway integrates various pro-proliferative and anti-apoptotic stimuli and is involved in regulatory T-cell (TREG) development. As these processes contribute to the pathogenesis of myelodysplastic syndromes (MDS), we hypothesized that mTOR modulation with temsirolimus (TEM) might show activity in MDS. This prospective multicentre trial enrolled lower and higher risk MDS patients, provided that they were transfusion-dependent/neutropenic or relapsed/refractory to 5-azacitidine, respectively. All patients received TEM at a weekly dose of 25 mg. Of the 9 lower- and 11 higher-risk patients included, only 4 (20%) reached the response assessment after 4 months of treatment and showed stable disease without haematological improvement. The remaining patients discontinued TEM prematurely due to adverse events. Median overall survival (OS) was not reached in the lower-risk group and 296 days in the higher-risk group. We observed a significant decline of bone marrow (BM) vascularisation (P = 0·006) but were unable to demonstrate a significant impact of TEM on the balance between TREG and pro-inflammatory T-helper-cell subsets within the peripheral blood or BM. We conclude that mTOR-modulation with TEM at a dose of 25 mg per week is accompanied by considerable toxicity and has no beneficial effects in elderly MDS patients. © 2016 John Wiley & Sons Ltd.

  5. SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

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    Ashok, Ajay [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288 (United States); Kanwar, Jagat Rakesh [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Krishnan, Uma Maheswari [Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology (SCBT), SASTRA University, Thanjavur 613401 (India); Kanwar, Rupinder Kaur, E-mail: rupinder.kanwar@deakin.edu.au [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia)

    2017-01-01

    Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxic microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic injury

  6. Prognostic role of hypoxia-inducible factor-1 alpha expression in osteosarcoma: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Ren HY

    2016-03-01

    Full Text Available Hai-Yong Ren,1 Yin-Hua Zhang,1,2 Heng-Yuan Li,1 Tao Xie,1 Ling-Ling Sun,1 Ting Zhu,1 Sheng-Dong Wang,1 Zhao-Ming Ye1 1Department of Orthopaedics, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 2The First Department of Orthopaedics, Hospital of Zhejiang General Corps of Armed Police Forces, Jiaxing, People’s Republic of China Background: Hypoxia-inducible factor-1α (HIF-1α plays an important role in tumor progression and metastasis. A number of studies have investigated the association of HIF-1α with prognosis and clinicopathological characteristics of osteosarcoma but yielded inconsistent results.  Method: Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs and odds ratios (ORs with corresponding confidence intervals (CIs were calculated to assess the prognostic value of HIF-1α expression. The standard mean difference was used to analyze the continuous variable.  Results: Finally, nine studies comprising 486 patients were subjected to final analysis. Protein expression level of HIF-1α was found to be significantly related to overall survival (HR =3.0; 95% CI: 1.46–6.15, disease-free survival (HR =2.23; 95% CI: 1.26–3.92, pathologic grade (OR =21.33; 95% CI: 4.60–98.88, tumor stage (OR =10.29; 95% CI: 3.55–29.82, chemotherapy response (OR =9.68; 95% CI: 1.87–50.18, metastasis (OR =5.06; 95% CI: 2.87–8.92, and microvessel density (standard mean difference =2.83; 95% CI: 2.28–3.39.  Conclusion: This meta-analysis revealed that overexpression of HIF-1α is a predictive factor of poor outcomes for osteosarcoma. HIF-1α appeared to play an important role in prognostic evaluation and may be a potential target in antitumoral therapy. Keywords: HIF-1α, osteosarcoma, prognosis, meta-analysis

  7. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

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    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  8. Rapamycin sensitizes T-ALL cells to dexamethasone-induced apoptosis

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    Mu Dezhi

    2010-11-01

    Full Text Available Abstract Background Glucocorticoid (GC resistance is frequently seen in acute lymphoblastic leukemia of T-cell lineage (T-ALL. In this study we investigate the potential and mechanism of using rapamycin to restore the sensitivity of GC-resistant T-ALL cells to dexamethasone (Dex treatment. Methods Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide (MTT assay. Fluorescence-activated cell sorting (FACS analysis was used to analyze apoptosis and cell cycles. Western blot analysis was performed to test the expression of the downstream effector proteins of mammalian target of rapamycin (mTOR, the cell cycle regulatory proteins, and apoptosis associated proteins. Results 10 nM rapamycin markedly increased GC sensitivity in GC-resistant T-ALL cells and this effect was mediated, at least in part, by inhibition of mTOR signaling pathway. Cell cycle arrest was associated with modulation of G1-S phase regulators. Both rapamycin and Dex can induce up-regulation of cyclin-dependent kinase (CDK inhibitors of p21 and p27 and co-treatment of rapamycin with Dex resulted in a synergistic induction of their expressions. Rapamycin did not obviously affect the expression of cyclin A, whereas Dex induced cyclin A expression. Rapamycin prevented Dex-induced expression of cyclin A. Rapamycin had a stronger inhibition of cyclin D1 expression than Dex. Rapamycin enhanced GC-induced apoptosis and this was not achieved by modulation of glucocorticoid receptor (GR expression, but synergistically up-regulation of pro-apoptotic proteins like caspase-3, Bax, and Bim, and down-regulation of anti-apoptotic protein of Mcl-1. Conclusion Our data suggests that rapamycin can effectively reverse GC resistance in T-ALL and this effect is achieved by inducing cell cycles arrested at G0/G1 phase and activating the intrinsic apoptotic program. Therefore, combination of mTOR inhibitor rapamycin with GC containing protocol might be an attracting

  9. Therapeutic effect of intra-articular injection of ribbon-type decoy oligonucleotides for hypoxia inducible factor-1 on joint contracture in an immobilized knee animal model.

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    Sotobayashi, Daisuke; Kawahata, Hirohisa; Anada, Natsuki; Ogihara, Toshio; Morishita, Ryuichi; Aoki, Motokuni

    2016-08-01

    Limited range of motion (ROM) as a result of joint contracture in treatment associated with joint immobilization or motor paralysis is a critical issue. However, its molecular mechanism has not been fully clarified and a therapeutic approach is not yet established. In the present study, we investigated its molecular mechanism, focusing on the role of a transcription factor, hypoxia inducible factor-1 (HIF-1), which regulates the expression of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), and evaluated the possibility of molecular therapy to inhibit HIF-1 activation by ribbon-type decoy oligonucleotides (ODNs) for HIF-1 using immobilized knee animal models. In a mouse model, ROM of the immobilized knee significantly decreased in a time-dependent manner, accompanied by synovial hypertrophy. Immunohistochemical studies suggested that CTGF and VEGF are implicated in synovial hypertrophy with fibrosis. CTGF and VEGF were up-regulated at both the mRNA and protein levels at 1 and 2 weeks after immobilization, subsequent to up-regulation of HIF-1 mRNA and transcriptional activation of HIF-1. Of importance, intra-articular transfection of decoy ODNs for HIF-1 in a rat model successfully inhibited transcriptional activation of HIF-1, followed by suppression of expression of CTGF and VEGF, resulting in attenuation of restricted ROM, whereas transfection of scrambled decoy ODNs did not. The present study demonstrates the important role of HIF-1 in the initial progression of immobilization-induced joint contracture, and indicates the possibility of molecular treatment to prevent the progression of joint contracture prior to intervention with physical therapy. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. MiR-142 modulates human pancreatic cancer proliferation and invasion by targeting hypoxia-inducible factor 1 (HIF-1α in the tumor microenvironments

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    Yebin Lu

    2017-02-01

    Full Text Available MicroRNAs regulate most protein-coding genes, including genes important in cancer and other diseases. In this study, we demonstrated that the expression of miR-142 could be significantly suppressed in pancreatic cancer specimens and cell lines compared to their adjacent tissues and normal pancreatic cells. Growth and invasion of PANC-1 and SW1990 cells were attenuated by overexpression of miR-142 in vitro. With the help of bioinformatics analysis, hypoxia-inducible factor 1 (HIF-1α was identified to be a direct target of miR-142, and a luciferase reporter experiment confirmed this discovery. Overexpression of miR-142 decreases protein expression of HIF-1α. In the hypoxic microenvironment, HIF-1α was up-regulated while miR-142 was down-regulated. The invaded cells significantly increased in the hypoxic microenvironment compared to the normoxic microenvironment. The hypoxia treatment induced cells’ proliferation, and invasion could be inhibited by miR-142 overexpression or HIF-1α inhibition. Moreover, expression of epithelial-mesenchymal transition (EMT markers, Vimentin, VEGF-C and E-cad, was altered under hypoxia conditions and regulated by miR-142/HIF-1α. Above all, these findings provided insights on the functional mechanism of miR-142, suggesting that the miR-142/HIF-1α axis may interfere with the proliferative and invasive properties of pancreatic cancer cells, and indicated that miR-142 could be a potential therapeutic target for pancreatic cancer.

  11. Histone demethylase JMJD1A promotes urinary bladder cancer progression by enhancing glycolysis through coactivation of hypoxia inducible factor 1α.

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    Wan, W; Peng, K; Li, M; Qin, L; Tong, Z; Yan, J; Shen, B; Yu, C

    2017-07-06

    High aerobic glycolysis not only provides energy to cancer cells, but also supports their anabolic growth. JMJD1A, a histone demethylase that specifically demethylates H3K9me1/2, is overexpressed in multiple cancers, including urinary bladder cancer (UBC). It is unclear whether JMJD1A could promote cancer cell growth through enhancing glycolysis. In this study, we found that downregulation of JMJD1A decreased UBC cell proliferation, colony formation and xenograft tumor growth. Knockdown of JMJD1A inhibited glycolysis by decreasing the expression of genes participated in glucose metabolism, including GLUT1, HK2, PGK1, PGM, LDHA and MCT4. Mechanistically, JMJD1A cooperated with hypoxia inducible factor 1α (HIF1α), an important transcription factor for glucose metabolism, to induce the glycolytic gene expression. JMJD1A was recruited to the promoter of glycolytic gene PGK1 to demethylate H3K9me2. However, the JMJD1A (H1120Y) mutant, which loses the demethylase activity, failed to cooperate with HIF1α to induce the glycolytic gene expression, and failed to demethylate H3K9me2 on PGK1 promoter, suggesting that the demethylase activity of JMJD1A is essential for its coactivation function for HIF1α. Inhibition of glycolysis through knocking down HIF1α or PGK1 decelerated JMJD1A-enhanced UBC cell growth. Consistent with these results, a positive correlation between JMJD1A and several key glycolytic genes in human UBC samples was established by analyzing a microarray-based gene expression profile. In conclusion, our study demonstrates that JMJD1A promotes UBC progression by enhancing glycolysis through coactivation of HIF1α, implicating that JMJD1A is a potential molecular target for UBC treatment.

  12. Synergistic antitumor activity of rapamycin and EF24 via increasing ROS for the treatment of gastric cancer

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    Weiqian Chen

    2016-12-01

    Full Text Available Mechanistic/mammalian target of rapamycin (mTOR has emerged as a new potential therapeutic target for gastric cancer. Rapamycin and rapamycin analogs are undergoing clinical trials and have produced clinical responses in a subgroup of cancer patients. However, monotherapy with rapamycin at safe dosage fails to induce cell apoptosis and tumor regression which has hampered its clinical application. This has led to the exploration of more effective combinatorial regimens to enhance the effectiveness of rapamycin. In our present study, we have investigated the combination of rapamycin and a reactive oxygen species (ROS inducer EF24 in gastric cancer. We show that rapamycin increases intracellular ROS levels and displays selective synergistic antitumor activity with EF24 in gastric cancer cells. This activity was mediated through the activation of c-Jun N terminal kinase and endoplasmic reticulum stress (ER pathways in cancer cells. We also show that inhibiting ROS accumulation reverses ER stress and prevents apoptosis induced by the combination of rapamycin and EF24. These mechanisms were confirmed using human gastric cancer xenografts in immunodeficient mice. Taken together, our work provides a novel therapeutic strategy for the treatment of gastric cancer. The work reveals that ROS generation could be an important target for the development of new combination therapies for cancer treatment.

  13. Morphological and functional alterations of the ductus arteriosus in a chicken model of hypoxia-induced fetal growth retardation.

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    Van der Sterren, Saskia; Agren, Pia; Zoer, Bea; Kessels, Lilian; Blanco, Carlos E; Villamor, Eduardo

    2009-03-01

    The hypoxic conditions in which children with intrauterine growth retardation (IUGR) develop are hypothesized to alter the development of the ductus arteriosus (DA). We aimed to evaluate the effects of in ovo hypoxia on chicken DA morphometry and reactivity. Hypoxia (15% O2 from day 6 to 19 of the 21-d incubation period) produced a reduction in the body mass of the 19-d fetuses and a shortening of right and left DAs. However, ductal lumen and media cross-sectional areas were not affected by hypoxia. The ductal contractions induced by oxygen, KCl, H2O2, 4-aminopyridine, and endothelin-1 were similar in control and hypoxic fetuses. In contrast, the DAs from the hypoxic fetuses showed increased contractile responses to norepinephrine and phenylephrine and impaired relaxations to acetylcholine, sodium nitroprusside, and isoproterenol. The relaxations induced by 8-Br-cGMP, forskolin, Y-27632, and hydroxyfasudil were not altered by chronic hypoxia. In conclusion, chronic in ovo hypoxia-induced growth retardation in fetal chickens and altered the response of the DA to adrenergic agonists and to endothelium-dependent and -independent relaxing agents. Our observations support the concept that prolonged patency of the DA in infants with IUGR may be partially related with hypoxia-induced changes in local vascular mechanisms.

  14. Punicalagin, a polyphenol in pomegranate juice, downregulates p53 and attenuates hypoxia-induced apoptosis in cultured human placental syncytiotrophoblasts.

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    Chen, Baosheng; Longtine, Mark S; Nelson, D Michael

    2013-11-15

    Oxidative stress is associated with placental dysfunction and suboptimal pregnancy outcomes. Therapeutic interventions to limit placental injury from oxidative stress are lacking. Punicalagin is an ellagitannin and a potent antioxidant in pomegranate juice. We showed that both pomegranate juice and punicalagin decrease oxidative stress and apoptosis in cultured syncytiotrophoblasts. p53 is involved in the oxidative stress-induced apoptosis in trophoblasts. We now test the hypothesis that punicalagin limits trophoblast injury in vitro by regulating the levels of p53. We examined the expression of p53, mouse double minute 2 homolog, p21, hypoxia-inducible factor (HIF) α, and selected members of the B cell lymphoma 2 (BCL2) family of proteins in cultured syncytiotrophoblasts exposed to ≤1% oxygen in the absence or presence of punicalagin. We found that punicalagin attenuated hypoxia-induced apoptosis in syncytiotrophoblasts, as quantified by levels of cleaved poly-ADP ribose polymerase. This protective effect was in part mediated by reduced p53 activity shown by decreased expression of p21, lower HIF1α expression, and limited activity of caspases 9 and 3. There was no change in expression of proteins in the BCL2 family, which are also important in apoptosis. The data support a role for downregulation of p53 in the protection of human trophoblasts by punicalagin.

  15. Blocking mTOR Signalling with Rapamycin Ameliorates Imiquimod-induced Psoriasis in Mice.

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    Bürger, Claudia; Shirsath, Nitesh; Lang, Victoria; Diehl, Sandra; Kaufmann, Roland; Weigert, Andreas; Han, Ying-Ying; Ringel, Christian; Wolf, Peter

    2017-10-02

    The mTOR (mechanistic target of rapamycin) inhibitor rapamycin has long been known for its immune suppressive properties, but it has shown limited therapeutic success when given systemically to patients with psoriasis. Recent data have shown that the mTOR pathway is hyperactivated in lesional psoriatic skin, which probably contributes to the disease by interfering with maturation of keratinocytes. This study investigated the effect of topical rapamycin treatment in an imiquimod-induced psoriatic mouse model. The disease was less severe if the mice had received rapamycin treatment. Immunohistological analysis revealed that rapamycin not only prevented the activation of mTOR signalling (P-mTOR and P-S6 levels), but almost normalized the expression of epidermal differentiation markers. In addition, the influx of innate immune cells into the draining lymph nodes was partially reduced by rapamycin treatment. These data emphasize the role of mTOR signalling in the pathogenesis of psoriasis, and support the investigation of topical mTOR inhibition as a novel anti-psoriatic strategy.

  16. Combinatorial Antitumor Effect of Rapamycin and β-Elemene in Follicular Thyroid Cancer Cells

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    Jun Zhou

    2016-01-01

    Full Text Available Background. mTOR signaling would be a promising target for thyroid cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in most cancer types was modest. A new focus on development of combinatorial strategies with rapalogs is increasing. Objective. Investigating the combinatorial antitumor effect of rapamycin and β-elemene in follicular thyroid cancer cells. Methods. MTT assay was used to determine the FTC-133 cell proliferation after culturing with rapamycin and/or β-elemene. To analyze their combinatorial effect, immunoblotting was performed to analyze the activation status of AKT. Moreover, β-elemene attenuated rapamycin-induced immunosuppression was tested in mice. Results. Combination of rapamycin and β-elemene exerted significant synergistic antiproliferative effects in FTC-133 cell lines in vitro, based on inhibiting the AKT feedback activation induced by rapamycin. In vivo, the β-elemene could attenuate rapamycin-induced immunosuppression via reversing imbalance of Treg/Th17, with the underlying mechanism needed to be declared. Conclusions. We demonstrate that the novel combination of mTOR inhibitor with β-elemene synergistically attenuates tumor cell growth in follicular thyroid cancer, which requires additional preclinical validation.

  17. Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation

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    Walpen, Thomas; Kalus, Ina [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Schwaller, Juerg [Department of Biomedicine, University of Basel, 4031 Basel (Switzerland); Peier, Martin A. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Battegay, Edouard J. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland); Humar, Rok, E-mail: Rok.Humar@usz.ch [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Pim1{sup -/-} endothelial cell proliferation displays increased sensitivity to rapamycin. Black-Right-Pointing-Pointer mTOR inhibition by rapamycin enhances PIM1 cytosolic and nuclear protein levels. Black-Right-Pointing-Pointer Truncation of Pim1 beyond serine 276 results in nuclear localization of the kinase. Black-Right-Pointing-Pointer Nuclear PIM1 increases endothelial proliferation independent of rapamycin. -- Abstract: The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1{sup -/-} cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation

  18. Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes

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    Bora E. Baysal

    2013-09-01

    Full Text Available Background. RNA editing is a post-transcriptional regulatory mechanism that can alter the coding sequences of certain genes in response to physiological demands. We previously identified C-to-U RNA editing (C136U, R46X which inactivates a small fraction of succinate dehydrogenase (SDH; mitochondrial complex II subunit B gene (SDHB mRNAs in normal steady-state peripheral blood mononuclear cells (PBMCs. SDH is a heterotetrameric tumor suppressor complex which when mutated causes paraganglioma tumors that are characterized by constitutive activation of hypoxia inducible pathways. Here, we studied regulation, extent and cell type origin of SDHB RNA editing.Methods. We used short-term cultured PBMCs obtained from random healthy platelet donors, performed monocyte enrichment by cold aggregation, employed a novel allele-specific quantitative PCR method, flow cytometry, immunologic cell separation, gene expression microarray, database analysis and high-throughput RNA sequencing.Results. While the editing rate is low in uncultured monocyte-enriched PBMCs (average rate 2.0%, range 0.4%–6.3%, n = 42, it is markedly upregulated upon exposure to 1% oxygen tension (average rate 18.2%, range 2.8%–49.4%, n = 14 and during normoxic macrophage differentiation in the presence of serum (average rate 10.1%, range 2.7%–18.8%, n = 17. The normoxic induction of SDHB RNA editing was associated with the development of dense adherent aggregates of monocytes in culture. CD14-positive monocyte isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures (n = 5 and 1.68-fold in hypoxic cultures (n = 4. CD14-negative lymphocytes showed no evidence of SDHB editing. The SDHB genomic DNA remained wild-type during increased RNA editing. Microarray analysis showed expression changes in wound healing and immune response pathway genes as the editing rate increased in normoxic cultures. High-throughput sequencing of SDHB and SDHD transcripts

  19. Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia-inducible factors HIF1α and HIF2α.

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    Schöning, Jennifer Petra; Monteiro, Michael; Gu, Wenyi

    2017-02-01

    Chemotherapy resistance is a major contributor to poor treatment responses and tumour relapse, the development of which has been strongly linked to the action of cancer stem cells (CSCs). Mounting evidence suggests that CSCs are reliant on low oxygen conditions and hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) to maintain their stem cell features. Research in the last decade has begun to clarify the functional differences between the two HIFα subtypes (HIFαs). Here, we review and discuss these differences in relation to CSC-associated drug resistance. Both HIFαs contribute to CSC survival but play different roles -HIF1α being more responsible for survival functions and HIF2α for stemness traits such as self-renewal - and are sensitive to different degrees of hypoxia. Failure to account for physiologically relevant oxygen concentrations in many studies may influence the current understanding of the roles of HIFαs. We also discuss how hypoxia and HIFαs contribute to CSC drug resistance via promotion of ABC drug transporters Breast cancer resistance protein (BCRP), MDR1, and MRP1 and through maintenance of quiescence. Additionally, we explore the PI3K/AKT cell survival pathway that may support refractory cancer by promoting CSCs and activating both HIF1α and HIF2α. Accordingly, HIF1α and HIF2α inhibition, potentially via PI3K/AKT inhibitors, could reduce chemotherapy resistance and prevent cancer relapse. © 2016 John Wiley & Sons Australia, Ltd.

  20. Reactive Oxygen Species-Reducing Strategies Improve Pulmonary Arterial Responses to Nitric Oxide in Piglets with Chronic Hypoxia-Induced Pulmonary Hypertension

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    Dikalova, Anna; Slaughter, James C.; Kaplowitz, M.R.; Zhang, Y.; Aschner, Judy L.

    2013-01-01

    Abstract Aims: There are no effective treatments for chronic pulmonary hypertension in infants with cardiopulmonary disorders associated with hypoxia, such as those with chronic lung disease. These patients often have poor or inconsistent pulmonary dilator responses to inhaled nitric oxide (iNO) therapy for unknown reasons. One possible explanation for poor responsiveness to iNO is reduced NO bioavailability caused by interactions between reactive oxygen species (ROS) and NO. Our major aim was to determine if strategies to reduce ROS improve dilator responses to the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), in resistance pulmonary arteries (PRAs) from a newborn piglet model of chronic pulmonary hypertension. Results: The dilation to SNAP was significantly impaired in PRAs from piglets with chronic hypoxia-induced pulmonary hypertension. ROS scavengers, including cell-permeable and impermeable agents to degrade hydrogen peroxide (H2O2), improved dilation to SNAP in PRAs from chronically hypoxic piglets. Treatment with agents to inhibit nitric oxide synthase and NADPH oxidase, potential enzymatic sources of ROS, also improved dilation to SNAP in PRAs from hypoxic piglets. Innovation: Our studies are the first to utilize a newborn model of chronic pulmonary hypertension to evaluate the impact of a number of potential therapeutic strategies for ROS removal on responses to exogenous NO in the vessels most relevant to the regulation of pulmonary vascular resistance (PRA). Conclusions: Strategies aimed at reducing ROS merit further evaluation and consideration as therapeutic approaches to improve responses to iNO in infants with chronic pulmonary hypertension. Antioxid. Redox Signal. 18, 1727–1738. PMID:23244497

  1. RNA interference targeting hypoxia-inducible factor 1α via a novel multifunctional surfactant attenuates glioma growth in an intracranial mouse model.

    Science.gov (United States)

    Gillespie, David L; Aguirre, Maria T; Ravichandran, Sandhya; Leishman, Lisa L; Berrondo, Claudia; Gamboa, Joseph T; Wang, Libo; King, Rose; Wang, Xuli; Tan, Mingqian; Malamas, Anthony; Lu, Zheng-Rong; Jensen, Randy L

    2015-02-01

    High-grade gliomas are the most common form of adult brain cancer, and patients have a dismal survival rate despite aggressive therapeutic measures. Intratumoral hypoxia is thought to be a main contributor to tumorigenesis and angiogenesis of these tumors. Because hypoxia-inducible factor 1α (HIF-1α) is the major mediator of hypoxia-regulated cellular control, inhibition of this transcription factor may reduce glioblastoma growth. Using an orthotopic mouse model with U87-LucNeo cells, the authors used RNA interference to knock down HIF-1α in vivo. The small interfering RNA (siRNA) was packaged using a novel multifunctional surfactant, 1-(aminoethyl) iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), a nucleic acid carrier that facilitates cellular uptake and intracellular release of siRNA. Stereotactic injection was used to deliver siRNA locally through a guide-screw system, and delivery/uptake was verified by imaging of fluorescently labeled siRNA. Osmotic pumps were used for extended siRNA delivery to model a commonly used human intracranial drug-delivery technique, convection-enhanced delivery. Mice receiving daily siRNA injections targeting HIF-1α had a 79% lower tumor volume after 50 days of treatment than the controls. Levels of the HIF-1 transcriptional targets vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), c-MET, and carbonic anhydrase-IX (CA-IX) and markers for cell growth (MIB-1 and microvascular density) were also significantly lower. Altering the carrier EHCO by adding polyethylene glycol significantly increased the efficacy of drug delivery and subsequent survival. Treating glioblastoma with siRNA targeting HIF-1α in vivo can significantly reduce tumor growth and increase survival in an intracranial mouse model, a finding that has direct clinical implications.

  2. Ascorbic acid, but not dehydroascorbic acid increases intracellular vitamin C content to decrease Hypoxia Inducible Factor -1 alpha activity and reduce malignant potential in human melanoma.

    Science.gov (United States)

    Fischer, Adam P; Miles, Sarah L

    2017-02-01

    Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α. HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with pascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. Inhibition of protein kinase C delta attenuates allergic airway inflammation through suppression of PI3K/Akt/mTOR/HIF-1 alpha/VEGF pathway.

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    Yun Ho Choi

    Full Text Available Vascular endothelial growth factor (VEGF is supposed to contribute to the pathogenesis of allergic airway disease. VEGF expression is regulated by a variety of stimuli such as nitric oxide, growth factors, and hypoxia-inducible factor-1 alpha (HIF-1α. Recently, inhibition of the mammalian target of rapamycin (mTOR has been shown to alleviate cardinal asthmatic features, including airway hyperresponsiveness, eosinophilic inflammation, and increased vascular permeability in asthma models. Based on these observations, we have investigated whether mTOR is associated with HIF-1α-mediated VEGF expression in allergic asthma. In studies with the mTOR inhibitor rapamycin, we have elucidated the stimulatory role of a mTOR-HIF-1α-VEGF axis in allergic response. Next, the mechanisms by which mTOR is activated to modulate this response have been evaluated. mTOR is known to be regulated by phosphoinositide 3-kinase (PI3K/Akt or protein kinase C-delta (PKC δ in various cell types. Consistent with these, our results have revealed that suppression of PKC δ by rottlerin leads to the inhibition of PI3K/Akt activity and the subsequent blockade of a mTOR-HIF-1α-VEGF module, thereby attenuating typical asthmatic attack in a murine model. Thus, the present data indicate that PKC δ is necessary for the modulation of the PI3K/Akt/mTOR signaling cascade, resulting in a tight regulation of HIF-1α activity and VEGF expression. In conclusion, PKC δ may represent a valuable target for innovative therapeutic treatment of allergic airway disease.

  4. E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells.

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    Gholizadeh, Shima; Visweswaran, Ganesh Ram R; Storm, Gert; Hennink, Wim E; Kamps, Jan A A M; Kok, Robbert J

    2017-10-13

    Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-α (TNF-α) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.;hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-α activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells. Copyright © 2017. Published by Elsevier B.V.

  5. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

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    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker; (Wyeth)

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  6. Rapamycin-Induced Apoptosis in HGF-Stimulated Lens Epithelial Cells by AKT/mTOR, ERK and JAK2/STAT3 Pathways

    Directory of Open Access Journals (Sweden)

    Fang Tian

    2014-08-01

    Full Text Available Hepatocyte growth factor (HGF induced the proliferation of lens epithelial cells (LECs and may be a major cause of posterior capsule opacification (PCO, which is the most frequent postoperative complication of cataract surgery. To date, several agents that can block LECs proliferation have been studied, but none have been used in clinic. Recently, accumulating evidence has suggested rapamycin, the inhibitor of mTOR (mammalian target of Rapamycin, was associated with the induction of apoptosis in LECs. The purpose of our study was to investigate the potential effects of rapamycin on HGF-induced LECs and the underlying mechanisms by which rapamycin exerted its actions. Using cell proliferation, cell viability and flow cytometric apoptosis assays, we found that rapamycin potently not only suppressed proliferation but also induced the apoptosis of LECs in a dose-dependent manner under HGF administration. Further investigation of the underlying mechanism using siRNA transfection revealed that rapamycin could promote apoptosis of LECs via inhibiting HGF-induced phosphorylation of AKT/mTOR, ERK and JAK2/STAT3 signaling molecules. Moreover, the forced expression of AKT, ERK and STAT3 could induce a significant suppression of apoptosis in these cells after treatment of rapamycin. Together, these findings suggested that rapamycin-induced apoptosis in HGF-stimulated LECs is accompanied by inhibition of AKT/mTOR, ERK and JAK2/STAT3 pathways, which supports its use to inhibit PCO in preclinical studies and provides theoretical foundation for future possible practice.

  7. Towards natural mimetics of metformin and rapamycin.

    Science.gov (United States)

    Aliper, Alexander; Jellen, Leslie; Cortese, Franco; Artemov, Artem; Karpinsky-Semper, Darla; Moskalev, Alexey; Swick, Andrew G; Zhavoronkov, Alex

    2017-11-15

    Aging is now at the forefront of major challenges faced globally, creating an immediate need for safe, widescale interventions to reduce the burden of chronic disease and extend human healthspan. Metformin and rapamycin are two FDA-approved mTOR inhibitors proposed for this purpose, exhibiting significant anti-cancer and anti-aging properties beyond their current clinical applications. However, each faces issues with approval for off-label, prophylactic use due to adverse effects. Here, we initiate an effort to identify nutraceuticals-safer, naturally-occurring compounds-that mimic the anti-aging effects of metformin and rapamycin without adverse effects. We applied several bioinformatic approaches and deep learning methods to the Library of Integrated Network-based Cellular Signatures (LINCS) dataset to map the gene- and pathway-level signatures of metformin and rapamycin and screen for matches among over 800 natural compounds. We then predicted the safety of each compound with an ensemble of deep neural network classifiers. The analysis revealed many novel candidate metformin and rapamycin mimetics, including allantoin and ginsenoside (metformin), epigallocatechin gallate and isoliquiritigenin (rapamycin), and withaferin A (both). Four relatively unexplored compounds also scored well with rapamycin. This work revealed promising candidates for future experimental validation while demonstrating the applications of powerful screening methods for this and similar endeavors.

  8. 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

  9. Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma.

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    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.

  10. Hypoxia Inducible Factor 1 (HIF1) Activation in U87 Glioma Cells Involves a Decrease in Reactive Oxygen Species Production and Protein Kinase C Activity

    Science.gov (United States)

    1998-06-29

    transcription factors NFkB, AP-l and Sox (Fandrey et al., 1994, Flohe et al., 1997, Hidalgo et al., 1997). Indeed, the bacterial transcription factor OxyR has...dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J Biol Chern 270: 21021-7. Flohe , L., R. Brigelius- Flohe , C

  11. Vascular endothelial growth factor and hypoxia-inducible factor-1α gene polymorphisms and coronary collateral formation in patients with coronary chronic total occlusions

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    Vincent Amoah

    2016-06-01

    Full Text Available Introduction: We evaluated the association between two single nucleotide polymorphisms of the vascular endothelial growth factor gene and one of the hypoxia-inducible factor-1α gene and the degree of coronary collateral formation in patients with a coronary chronic total occlusion. Methods: Totally, 98 patients with symptomatic coronary artery disease and a chronic total occlusion observed during coronary angiography were recruited. Genotyping of two vascular endothelial growth factor promoter single nucleotide polymorphisms (−152G>A and −165C>T and the C1772T single nucleotide polymorphism of hypoxia-inducible factor-1α were performed using polymerase chain reaction and restriction fragment length polymorphism analysis. The presence and extent of collateral vessel filling was scored by blinded observers using the Rentrop grade. Results: We found no association between the vascular endothelial growth factor −152G>A, −165C>T and hypoxia-inducible factor-1α −1772C>T with the presence and filling of coronary collateral vessels. A history of percutaneous coronary intervention and transient ischaemic attack/cerebrovascular accident were associated with the presence of enhanced collateral vessel formation following binary logistic regression analysis. Conclusion: The study findings suggest that coronary collateral formation is not associated with the tested polymorphic variants of vascular endothelial growth factor and hypoxia-inducible factor-1α in patients with symptomatic coronary artery disease and the presence of a chronic total occlusion.

  12. Hypoxia-inducible Lipid Droplet-associated (HILPDA) Is a Novel Peroxisome Proliferator-activated Receptor (PPAR) Target Involved in Hepatic Triglyceride Secretion

    NARCIS (Netherlands)

    Mattijsen, F.; Georgiadi, A.; Andasarie, T.; Szalowska, E.; Zota, A.; Krones-Herzig, A.; Kersten, A.H.

    2014-01-01

    Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a

  13. Constitutive expression of hypoxia-inducible factor-1 α in keratinocytes during the repair of skin wounds in horses.

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    Deschene, Karine; Céleste, Christophe; Boerboom, Derek; Theoret, Christine L

    2011-01-01

    As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia-inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (pwounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (pequine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type. © 2011 by the Wound Healing Society.

  14. 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.

  15. mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy.

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    Srivastava, Isha N; Shperdheja, Jona; Baybis, Marianna; Ferguson, Tanya; Crino, Peter B

    2016-01-01

    Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in

  16. Efficacy of aqueous extract of Hippophae rhamnoides and its bio-active flavonoids against hypoxia-induced cell death.

    Science.gov (United States)

    Tulsawani, Rajkumar; Gupta, Rashmi; Misra, Kshipra

    2013-01-01

    To investigate the protective efficacy of aqueous extract of Hippophae rhamnoides against chronic hypoxic injury using primary rat hepatocytes. The extract was prepared using maceration method and characterized by its phenolic and flavonoid content and chemical antioxidant capacity using ferric reducing antioxidant power assay. Hepatocytes were maintained in hypoxia chamber (3% and 1% oxygen) for 72 h. The cells kept under normoxic condition served as control. The cells were treated with the extract and flavonoids; isorhamentin, kaempferol or qurecetin-3-galactoside. After the end of exposure period; cell survival, reactive oxygen species (ROS), leakage of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were measured. The extract showed presence of high phenolic and flavonoid content with significant antioxidant activity in chemical assay. The cell exposed to hypoxia showed concentration dependent cell death and harbored higher reactive oxygen species. In addition, these cells showed significant leakage of intracellular LDH, ALT, and AST accompanied by the diminished levels/activities of GSH, GPx, and SOD. The treatment of cells with aqueous extract of H. rhamnoides reduced hypoxia-induced cell death and prevented increase in ROS levels and leakage of intracellular LDH, ALT, and AST from cells. Moreover, these cells maintained better levels/activities of GSH, GPx, and SOD in comparison to the respective controls. The major flavonoids present in aqueous extract of H. rhamnoides; quercetin-3-galactoside, kaempferol, and isorhamentin also prevented hypoxia induced cell injury individually or in combination, however, the protection offered by these compounds taken together could not match to that of the extract. Overall the findings reveal significance of aqueous extract of H. rhamnoides in controlling ROS-meditated hypoxic

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

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    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.

  18. Hypoxia-induced miR-15a promotes mesenchymal ablation and adaptation to hypoxia during lung development in chicken.

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    Rui Hao

    Full Text Available The lungs undergo changes that are adaptive for high elevation in certain animal species. In chickens, animals bred at high elevations (e.g., Tibet chickens are better able to hatch and survive under high-altitude conditions. In addition, lowland chicken breeds undergo physiological effects and suffer greater mortality when they are exposed to hypoxic conditions during embryonic development. Although these physiological effects have been noted, the mechanisms that are responsible for hypoxia-induced changes in lung development and function are not known. Here we have examined the role of a particular microRNA (miRNA in the regulation of lung development under hypoxic conditions. When chicks were incubated in low oxygen (hypoxia, miR-15a was significantly increased in embryonic lung tissue. The expression level of miR-15a in hypoxic Tibet chicken embryos increased and remained relatively high at embryonic day (E16-20, whereas in normal chickens, expression increased and peaked at E19-20, at which time the cross-current gas exchange system (CCGS is developing. Bcl-2 was a translationally repressed target of miR-15a in these chickens. miR-16, a cluster and family member of miR-15a, was detected but did not participate in the posttranscriptional regulation of bcl-2. Around E19, the hypoxia-induced decrease in Bcl-2 protein resulted in apoptosis in the mesenchyme around the migrating tubes, which led to an expansion and migration of the tubes that would become the air capillary network and the CCGS. Thus, interfering with miR-15a expression in lung tissue may be a novel therapeutic strategy for hypoxia insults and altitude adaptation.

  19. Hypoxia-Induced miR-15a Promotes Mesenchymal Ablation and Adaptation to Hypoxia during Lung Development in Chicken

    Science.gov (United States)

    Hao, Rui; Hu, Xiaoxiang; Wu, Changxin; Li, Ning

    2014-01-01

    The lungs undergo changes that are adaptive for high elevation in certain animal species. In chickens, animals bred at high elevations (e.g., Tibet chickens) are better able to hatch and survive under high-altitude conditions. In addition, lowland chicken breeds undergo physiological effects and suffer greater mortality when they are exposed to hypoxic conditions during embryonic development. Although these physiological effects have been noted, the mechanisms that are responsible for hypoxia-induced changes in lung development and function are not known. Here we have examined the role of a particular microRNA (miRNA) in the regulation of lung development under hypoxic conditions. When chicks were incubated in low oxygen (hypoxia), miR-15a was significantly increased in embryonic lung tissue. The expression level of miR-15a in hypoxic Tibet chicken embryos increased and remained relatively high at embryonic day (E)16–20, whereas in normal chickens, expression increased and peaked at E19–20, at which time the cross-current gas exchange system (CCGS) is developing. Bcl-2 was a translationally repressed target of miR-15a in these chickens. miR-16, a cluster and family member of miR-15a, was detected but did not participate in the posttranscriptional regulation of bcl-2. Around E19, the hypoxia-induced decrease in Bcl-2 protein resulted in apoptosis in the mesenchyme around the migrating tubes, which led to an expansion and migration of the tubes that would become the air capillary network and the CCGS. Thus, interfering with miR-15a expression in lung tissue may be a novel therapeutic strategy for hypoxia insults and altitude adaptation. PMID:24887070

  20. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  1. Coronary Serum Obtained After Myocardial Infarction Induces Angiogenesis and Microvascular Obstruction Repair. Role of Hypoxia-inducible Factor-1A.

    Science.gov (United States)

    Ríos-Navarro, César; Hueso, Luisa; Miñana, Gema; Núñez, Julio; Ruiz-Saurí, Amparo; Sanz, María Jesús; Cànoves, Joaquin; Chorro, Francisco J; Piqueras, Laura; Bodí, Vicente

    2017-07-24

    Microvascular obstruction (MVO) exerts deleterious effects following acute myocardial infarction (AMI). We investigated coronary angiogenesis induced by coronary serum and the role of hypoxia-inducible factor-1A (HIF-1A) in MVO repair. Myocardial infarction was induced in swine by transitory 90-minute coronary occlusion. The pigs were divided into a control group and 4 AMI groups: no reperfusion, 1minute, 1 week and 1 month after reperfusion. Microvascular obstruction and microvessel density were quantified. The proangiogenic effect of coronary serum drawn from coronary sinus on endothelial cells was evaluated using an in vitro tubulogenesis assay. Circulating and myocardial HIF-1A levels and the effect of in vitro blockade of HIF-1A was assessed. Compared with control myocardium, microvessel density decreased at 90-minute ischemia, and MVO first occurred at 1minute after reperfusion. Both peaked at 1 week and almost completely resolved at 1 month. Coronary serum exerted a neoangiogenic effect on coronary endothelial cells in vitro, peaking at ischemia and 1minute postreperfusion (32 ± 4 and 41 ± 9 tubes vs control: 3 ± 3 tubes; P < .01). Hypoxia-inducible factor-1A increased in serum during ischemia (5-minute ischemia: 273 ± 52 pg/mL vs control: 148 ± 48 pg/mL; P < .01) being present on microvessels of all AMI groups (no reperfusion: 67% ± 5% vs control: 15% ± 17%; P < .01). In vitro blockade of HIF-1A reduced the angiogenic response induced by serum. Coronary serum represents a potent neoangiogenic stimulus even before reperfusion; HIF-1A might be crucial. Coronary neoangiogenesis induced by coronary serum can contribute to understanding the pathophysiology of AMI. Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  2. Multi-Vitamin B Supplementation Reverses Hypoxia-Induced Tau Hyperphosphorylation and Improves Memory Function in Adult Mice.

    Science.gov (United States)

    Yu, Lixia; Chen, Yuan; Wang, Weiguang; Xiao, Zhonghai; Hong, Yan

    2016-08-04

    Hypobaric hypoxia (HH) leads to reduced oxygen delivery to brain. It could trigger cognitive dysfunction and increase the risk of dementia including Alzheimer's disease (AD). The present study was undertaken in order to examine whether B vitamins (B6, B12, folate, and choline) could exert protective effects on hypoxia-induced memory deficit and AD related molecular events in mice. Adult male Kunming mice were assigned to five groups: normoxic control, hypoxic model (HH), hypoxia+vitamin B6/B12/folate (HB), hypoxia+choline (HC), hypoxia+vitamin B6/B12/folate+choline (HBC). Mice in the hypoxia, HB, HC, and HBC groups were exposed to hypobaric hypoxia for 8 h/day for 28 days in a decompression chamber mimicking 5500 meters of high altitude. Spatial and passive memories were assessed by radial arm and step-through passive test, respectively. Levels of tau and glycogen synthase kinase (GSK)-3β phosphorylation were detected by western blot. Homocysteine (Hcy) concentrations were determined using enzymatic cycling assay. Mice in the HH group exhibited significant spatial working and passive memory impairment, increased tau phosphorylation at Thr181, Ser262, Ser202/Thr205, and Ser396 in the cortex and hippocampus, and elevated Hcy levels compared with controls. Concomitantly, the levels of Ser9-phosphorylated GSK-3β were significantly decreased in brain after hypoxic treatment. Supplementations of vitamin B6/B12/folate+choline could significantly ameliorate the hypoxia-induced memory deficits, observably decreased Hcy concentrations in serum, and markedly attenuated tau hyperphosphorylation at multiple AD-related sites through upregulating inhibitory Ser9-phosphorylated GSK-3β. Our finding give further insight into combined neuroprotective effects of vitamin B6, B12, folate, and choline on brain against hypoxia.

  3. ATR controls cellular adaptation to hypoxia through positive regulation of hypoxia-inducible factor 1 (HIF-1) expression.

    Science.gov (United States)

    Fallone, F; Britton, S; Nieto, L; Salles, B; Muller, C

    2013-09-12

    Tumor cells adaptation to severe oxygen deprivation (hypoxia) plays a major role in tumor progression. The transcription factor HIF-1 (hypoxia-inducible factor 1), whose α-subunit is stabilized under hypoxic conditions is a key component of this process. Recent studies showed that two members of the phosphoinositide 3-kinase-related kinases (PIKKs) family, ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), regulate the hypoxic-dependent accumulation of HIF-1. These proteins initiate cellular stress responses when DNA damage occurs. In addition, it has been demonstrated that extreme hypoxia induces a replicative stress resulting in regions of single-stranded DNA at stalled replication forks and the activation of ATR (ataxia telangiectasia and Rad3 related protein), another member of the PIKKs family. Here, we show that even less severe hypoxia (0.1% O2) also induces activation of ATR through replicative stress. Importantly, in using either transiently silenced ATR cells, cells expressing an inactive form of ATR or cells exposed to an ATR inhibitor (CGK733), we demonstrate that hypoxic ATR activation positively regulates the key transcription factor HIF-1 independently of the checkpoint kinase Chk1. We show that ATR kinase activity regulates HIF-1α at the translational level and we find that the elements necessary for the regulation of HIF-1α translation are located within the coding region of HIF-1α mRNA. Finally, by using three independent cellular models, we clearly show that the loss of ATR expression and/or kinase activity results in the decrease of HIF-1 DNA binding under hypoxia and consequently affects protein expression levels of two HIF-1 target genes, GLUT-1 and CAIX. Taken together, our data show a new function for ATR in cellular adaptation to hypoxia through regulation of HIF-1α translation. Our work offers new prospect for cancer therapy using ATR inhibitors with the potential to decrease cellular adaptation in hypoxic

  4. Proteomic analysis of signaling network regulation in renal cell carcinomas with differential hypoxia-inducible factor-2α expression.

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    Lokesh Dalasanur Nagaprashantha

    Full Text Available BACKGROUND: The loss of von Hippel-Lindau (VHL protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α instead of hypoxia-inducible factor-1α (HIF1α. METHODS AND PRINCIPAL FINDINGS: We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography-mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335. CONCLUSIONS AND SIGNIFICANCE: Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.

  5. Altered expression of hypoxia-inducible factor-1α (HIF-1α and its regulatory genes in gastric cancer tissues.

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    Jihan Wang

    Full Text Available Tissue hypoxia induces reprogramming of cell metabolism and may result in normal cell transformation and cancer progression. Hypoxia-inducible factor 1-alpha (HIF-1α, the key transcription factor, plays an important role in gastric cancer development and progression. This study aimed to investigate the underlying regulatory signaling pathway in gastric cancer using gastric cancer tissue specimens. The integration of gene expression profile and transcriptional regulatory element database (TRED was pursued to identify HIF-1α ↔ NFκB1 → BRCA1 → STAT3 ← STAT1 gene pathways and their regulated genes. The data showed that there were 82 differentially expressed genes that could be regulated by these five transcription factors in gastric cancer tissues and these genes formed 95 regulation modes, among which seven genes (MMP1, TIMP1, TLR2, FCGR3A, IRF1, FAS, and TFF3 were hub molecules that are regulated at least by two of these five transcription factors simultaneously and were associated with hypoxia, inflammation, and immune disorder. Real-Time PCR and western blot showed increasing of HIF-1α in mRNA and protein levels as well as TIMP1, TFF3 in mRNA levels in gastric cancer tissues. The data are the first study to demonstrate HIF-1α-regulated transcription factors and their corresponding network genes in gastric cancer. Further study with a larger sample size and more functional experiments is needed to confirm these data and then translate into clinical biomarker discovery and treatment strategy for gastric cancer.

  6. Transarterial embolization combined with RNA interference targeting hypoxia-inducible factor-1α for hepatocellular carcinoma: a preliminary study of rat model.

    Science.gov (United States)

    Ni, Jia-Yan; Xu, Lin-Feng; Wang, Wei-Dong; Huang, Qiao-Sheng; Sun, Hong-Liang; Chen, Yao-Ting

    2017-02-01

    To study whether transarterial embolization (TAE) with RNA interference (RNAi) targeting hypoxia-inducible factor-1α (HIF-1α) can improve efficacy of TAE in treating hepatocellular carcinoma (HCC). CBRH-7919 rat hepatoma cell line was used and HCC models of rats were constructed. The siRNA transfection compound was made by mixing specific siRNA and Lipofectamine 2000™. Delivery and transfection of siRNA were administered by injecting iodized oil emulsion (diluted lipiodol and siRNA) via hepatic artery. The expression levels of mRNA and protein were detected using the real-time reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and western blotting assays, respectively. In vitro experiment, the specific HIF-1α-siRNA was proved to inhibit expression levels of HIF-1α and vascular endothelial growth factor (VEGF) effectively. In animal study, real-time RT-PCR assay showed the average relative mRNA expressions of HIF-1α were 0.31 ± 0.01, 0.65 ± 0.03, 0.46 ± 0.005, and 1.00 ± 0.00 in TAE + siRNA, siRNA, TAE, and control groups, respectively. Western blotting assay showed the average relative protein expressions of HIF-1α were 0.13 ± 0.02, 0.87 ± 0.02, 0.39 ± 0.02, and 1.02 ± 0.01 in TAE + siRNA, siRNA, TAE, and control groups, respectively. Compared with control, TAE, and siRNA groups, TAE + siRNA can significantly inhibit protein expressions of HIF-1α and VEGF (P HIF-1α < 0.001; P VEGF < 0.001). Overall survival of rats underwent TAE + siRNA was significantly longer than that of rats treated with TAE monotherapy (P = 0.001). This animal study showed TAE combined with HIF-1α-RNAi could significantly improve efficacy of TAE in treating HCC by inhibiting expressions of HIF-1α and VEGF after TAE treatment.

  7. The anti-proliferative effect of L-carnosine correlates with a decreased expression of hypoxia inducible factor 1 alpha in human colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Barbara Iovine

    Full Text Available In recent years considerable attention has been given to the use of natural substances as anticancer drugs. The natural antioxidant dipeptide L-carnosine belongs to this class of molecules because it has been proved to have a significant anticancer activity both in vitro and in vivo. Previous studies have shown that L-carnosine inhibits the proliferation of human colorectal carcinoma cells by affecting the ATP and Reactive Oxygen Species (ROS production. In the present study we identified the Hypoxia-Inducible Factor 1α (HIF-1α as a possible target of L-carnosine in HCT-116 cell line. HIF-1α protein is over-expressed in multiple types of human cancer and is the major cause of resistance to drugs and radiation in solid tumours. Of particular interest are experimental data supporting the concept that generation of ROS provides a redox signal for HIF-1α induction, and it is known that some antioxidants are able to suppress tumorigenesis by inhibiting HIF-1α. In the current study we found that L-carnosine reduces the HIF-1α protein level affecting its stability and decreases the HIF-1 transcriptional activity. In addition, we demonstrated that L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1α degradation. Finally, we compared the antioxidant activity of L-carnosine with that of two synthetic anti-oxidant bis-diaminotriazoles (namely 1 and 2, respectively. Despite these three compounds have the same ability in reducing intracellular ROS, 1 and 2 are more potent scavengers and have no effect on HIF-1α expression and cancer cell proliferation. These findings suggest that an analysis of L-carnosine antioxidant pathway will clarify the mechanism underlying the anti-proliferative effects of this dipeptide on colon cancer cells. However, although the molecular mechanism by which L-carnosine down regulates or inhibits the HIF-1α activity has not been yet elucidated, this ability may be promising in treating hypoxia

  8. GSK-3/Rb12 Pathway as a Novel Target of Rapamycin in Prostate Cancer

    Science.gov (United States)

    2005-11-01

    that also have a potent tumor suppressor effect. These drugs are currently being evaluated in clinical trials to treat human cancers including...tumors to inhibition of FRAP /mTOR." Proc Natl Acad Sci U S A 98(18): 10314-9. Noh, W. C., W. H. Mondesire, et al. (2004). "Determinants of rapamycin

  9. Rapamycin prevents seizures after depletion of STRADA in a rare neurodevelopmental disorder.

    Science.gov (United States)

    Parker, Whitney E; Orlova, Ksenia A; Parker, William H; Birnbaum, Jacqueline F; Krymskaya, Vera P; Goncharov, Dmitry A; Baybis, Marianna; Helfferich, Jelte; Okochi, Kei; Strauss, Kevin A; Crino, Peter B

    2013-04-24

    A rare neurodevelopmental disorder in the Old Order Mennonite population called PMSE (polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome; also called Pretzel syndrome) is characterized by infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter and subependymal regions. PMSE is caused by a homozygous deletion of exons 9 to 13 of the LYK5/STRADA gene, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin complex 1 (mTORC1). We show that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADA depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671 (p70S6Ki). We demonstrate that rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADA depletion in the mouse cerebral cortex. Constitutive mTORC1 signaling and a migration defect observed in fibroblasts from patients with PMSE were also prevented by mTORC1 inhibition. On the basis of these preclinical findings, we treated five PMSE patients with sirolimus (rapamycin) without complication and observed a reduction in seizure frequency and an improvement in receptive language. Our findings demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition may be a potential treatment for PMSE as well as other mTOR-associated neurodevelopmental disorders.

  10. IGF-1 attenuates hypoxia-induced atrophy but inhibits myoglobin expression in C2C12 skeletal muscle myotubes

    NARCIS (Netherlands)

    Peters, Eva L.; van der Linde, Sandra M.; Vogel, Ilse S.P.; Haroon, Mohammad; Offringa, Carla; de Wit, Gerard M.J.; Koolwijk, Pieter; van der Laarse, Willem J.; Jaspers, Richard T.

    2017-01-01

    Chronic hypoxia is associated with muscle wasting and decreased oxidative capacity. By contrast, training under hypoxia may enhance hypertrophy and increase oxidative capacity as well as oxygen transport to the mitochondria, by increasing myoglobin (Mb) expression. The latter may be a feasible

  11. Topical application of rapamycin ointment ameliorates Dermatophagoides farina body extract-induced atopic dermatitis in NC/Nga mice.

    Science.gov (United States)

    Yang, Fei; Tanaka, Mari; Wataya-Kaneda, Mari; Yang, Lingli; Nakamura, Ayumi; Matsumoto, Shoji; Attia, Mostafa; Murota, Hiroyuki; Katayama, Ichiro

    2014-08-01

    Atopic dermatitis (AD), a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. Recently, rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, has been reported to play a critical role in immune responses and has emerged as an effective immunosuppressive drug. In this study, we assessed whether inhibition of mTOR signalling could suppress dermatitis in mice. Rapamycin was topically applied to inflamed skin in a murine AD model that was developed by repeated topical application of Dermatophagoides farina body (Dfb) extract antigen twice weekly for 7 weeks in NC/Nga mice. The efficacy of topical rapamycin treatment was evaluated immunologically and serologically. Topical application of rapamycin reduced inflammatory cell infiltration in the dermis, alleviated the increase of serum IgE levels and resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. In addition, increased mTOR phosphorylation in the lesional skin was observed in our murine AD model. Topical application of rapamycin ointment inhibited Dfb antigen-induced dermatitis in NC/Nga mice, promising a new therapy for atopic dermatitis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats.

    NARCIS (Netherlands)

    Drion, C.M.; Borm, L.E.; Kooijman, L.; Aronica, E.; Wadman, W.J.; Hartog, A.F.; van Vliet, E.A.; Gorter, J.A.

    2016-01-01

    OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment

  13. Effects of rapamycin treatment after controlled cortical impact injury on neurogenesis and synaptic reorganization in the mouse dentate gyrus

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    Corwin R Butler

    2015-11-01

    Full Text Available Post-traumatic epilepsy (PTE is one consequence of traumatic brain injury (TBI. A prominent cell signaling pathway activated in animal models of both TBI and epilepsy is the mammalian target of rapamycin (mTOR. Inhibition of mTOR with rapamycin has shown promise as a potential modulator of epileptogenesis in several animal models of epilepsy, but cellular mechanisms linking mTOR expression and epileptogenesis are unclear. In this study, the role of mTOR in modifying functional hippocampal circuit reorganization after focal TBI induced by controlled cortical impact was investigated. Rapamycin (3 or 10 mg/kg, an inhibitor of mTOR signaling, was administered by intraperitoneal injection beginning on the day of injury and continued daily until tissue collection. Relative to controls, rapamycin treatment reduced dentate granule cell area in the hemisphere ipsilateral to the injury two weeks post-injury. Brain injury resulted in a significant increase in doublecortin immunolabeling in the dentate gyrus ipsilateral to the injury, indicating increased neurogenesis shortly after TBI. Rapamycin treatment prevented the increase in doublecortin labeling, with no overall effect on Fluoro-Jade B staining in the ipsilateral hemisphere, suggesting that rapamycin treatment reduced posttraumatic neurogenesis but did not prevent cell loss after injury. At later times post-injury (8-13 weeks, evidence of mossy fiber sprouting and increased recurrent excitation of dentate granule cells was detected, which were attenuated by rapamycin treatment. Rapamycin treatment also diminished seizure prevalence relative to vehicle-treated controls after TBI. Collectively, these results support a role for adult neurogenesis in PTE development and suggest that suppression of epileptogenesis by mTOR inhibition includes effects on post-injury neurogenesis.

  14. A critical role of interleukin-10 in modulating hypoxia-induced preeclampsia-like disease in mice.

    Science.gov (United States)

    Lai, Zhongbin; Kalkunte, Satyan; Sharma, Surendra

    2011-03-01

    Hypoxia has been implicated in the pathogenesis of preeclampsia, a hypertensive disorder of pregnancy. However, in vivo evidence and mechanistic understanding remain elusive. Preeclampsia is associated with impaired placental angiogenesis. We have recently shown that interleukin (IL)-10 can support trophoblast-driven endovascular crosstalk. Accordingly, we hypothesize that pathological levels of oxygen coupled with IL-10 deficiency induce severe preeclampsia-like features coupled with elevated production of antiangiogenic factors, apoptotic pathways, and placental injury. Exposure of pregnant wild-type and IL-10(-/-) mice to 9.5% oxygen resulted in graded placental injury and systemic symptoms of renal pathology, proteinuria (wild-type 645.15 ± 115.73 versus 198.09 ± 93.45; IL-10(-/-) 819.31 ± 127.85 versus 221.45 ± 82.73 μg/mg/24 hours) and hypertension (wild-type 118.37 ± 14.45 versus 78.67 ± 14.07; IL-10(-/-) 136.03 ± 22.59 versus 83.97 ± 18.25 mm Hg). Recombinant IL-10 reversed hypoxia-induced features in pregnant IL-10(-/-) mice confirming the protective role of IL-10 in preeclampsia. Hypoxic exposure caused marked elevation of soluble fms-like tyrosine kinase 1 (110.8 ± 20.1 versus 44.7 ± 11.9 ng/mL) in IL-10(-/-) mice compared with their wild-type counterparts (81.6 ± 13.1 versus 41.2 ± 8.9 ng/mL), whereas soluble endoglin was induced to similar levels in both strains (approximately 380 ± 50 versus 180 ± 31 ng/mL). Hypoxia-induced elevation of p53 was associated with marked induction of proapoptotic protein Bax, downregulation of Bcl-2, and trophoblast-specific apoptosis in utero-placental tissue. Collectively, we conclude that severe preeclampsia pathology could be triggered under certain threshold oxygen levels coupled with intrinsic IL-10 deficiency, which lead to excessive activation of antiangiogenic and apoptotic pathways.

  15. Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of); Park, Jong-Wan, E-mail: parkjw@snu.ac.kr [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

  16. Hypoxia inducible factor 3α plays a critical role in alveolarization and distal epithelial cell differentiation during mouse lung development.

    Directory of Open Access Journals (Sweden)

    Yadi Huang

    Full Text Available Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF. HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS or weak transcriptional activators (HIF3α/NEPAS. Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel

  17. Rapamycin enhances docetaxel-induced cytotoxicity in a androgen-independent prostate cancer xenograft model by survivin downregulation

    Energy Technology Data Exchange (ETDEWEB)

    Morikawa, Yasuyuki, E-mail: yasu-m@med.gunma-u.ac.jp [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan); Koike, Hidekazu; Sekine, Yoshitaka; Matsui, Hiroshi; Shibata, Yasuhiro; Ito, Kazuto; Suzuki, Kazuhiro [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Rapamycin (RPM) enhances the susceptibility of PC3 cells to docetaxel. Black-Right-Pointing-Pointer Low-dosage of docetaxel (DTX) did not reduce survivin expression levels in PC3 cells. Black-Right-Pointing-Pointer Combination treatment of RPM with DTX suppressed the expression of surviving. Black-Right-Pointing-Pointer SiRNA against survivin enhanced the susceptibility of PC3 cells to DTX. Black-Right-Pointing-Pointer RPM and DTX cotreatment inhibited PC3 cell growth and decreased surviving in vivo. -- Abstract: Background: Docetaxel is a first-line treatment choice in castration-resistant prostate cancer (CRPC). However, the management of CRPC remains an important challenge in oncology. There have been many reports on the effects of rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR), in the treatment of carcinogenesis. We assessed the cytotoxic effects of the combination treatment of docetaxel and rapamycin in prostate cancer cells. Furthermore, we examined the relationship between these treatments and survivin, which is a member of the inhibitory apoptosis family. Methods: Prostate cancer cells were cultured and treated with docetaxel and rapamycin. The effects on proliferation were evaluated with the MTS assay. In addition, we evaluated the effect on proliferation of the combination treatment induced knockdown of survivin expression by small interfering RNA transfection and docetaxel. Protein expression levels were assayed using western blotting. PC3 cells and xenograft growth in nude mice were used to evaluate the in vivo efficacy of docetaxel and its combination with rapamycin. Results: In vitro and in vivo, the combination of rapamycin with docetaxel resulted in a greater inhibition of proliferation than treatment with rapamycin or docetaxel alone. In addition, in vitro and in vivo, rapamycin decreased basal surviving levels, and cotreatment with docetaxel further decreased these levels

  18. Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2

    Directory of Open Access Journals (Sweden)

    Lan eYe

    2012-09-01

    Full Text Available Rapamycin, an inhibitor of mTOR complex 1 (mTORC1, improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24-48 hours. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

  19. Rapamycin Maintains the Chondrocytic Phenotype and Interferes with Inflammatory Cytokine Induced Processes

    Directory of Open Access Journals (Sweden)

    Andrea De Luna-Preitschopf

    2017-07-01

    Full Text Available Osteoarthritis (OA is hallmarked by a progressive degradation of articular cartilage. Besides risk factors including trauma, obesity or genetic predisposition, inflammation has a major impact on the development of this chronic disease. During the course of inflammation, cytokines such as tumor necrosis factor-alpha(TNF-α and interleukin (IL-1β are secreted by activated chondrocytes as well as synovial cells and stimulate the production of other inflammatory cytokines and matrix degrading enzymes. The mTORC1 inhibitor rapamycin is a clinical approved immunosuppressant and several studies also verified its chondroprotective effects in OA. However, the effect of blocking the mechanistic target of rapamycin complex (mTORC1 on the inflammatory status within OA is not well studied. Therefore, we aimed to investigate if inhibition of mTORC1 by rapamycin can preserve and sustain chondrocytes in an inflammatory environment. Patient-derived chondrocytes were cultured in media supplemented with or without the mTORC1 inhibitor rapamycin. To establish an inflammatory environment, either TNF-α or IL-1β was added to the media (=OA-model. The chondroprotective and anti-inflammatory effects of rapamycin were evaluated using sulfated glycosaminoglycan (sGAG release assay, Caspase 3/7 activity assay, lactate dehydrogenase (LDH assay and quantitative real time polymerase chain reaction (PCR. Blocking mTORC1 by rapamycin reduced the release and therefore degradation of sGAGs, which are components of the extracellular matrix secreted by chondrocytes. Furthermore, blocking mTORC1 in OA chondrocytes resulted in an enhanced expression of the main chondrogenic markers. Rapamycin was able to protect chondrocytes from cell death in an OA-model shown by reduced Caspase 3/7 activity and diminished LDH release. Furthermore, inhibition of mTORC1 preserved the chondrogenic phenotype of OA chondrocytes, but also reduced inflammatory processes within the OA-model. This study

  20. The hypoxia-inducible factor-responsive proteins semaphorin 4D and vascular endothelial growth factor promote tumor growth and angiogenesis in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Hua; Yang, Ying-Hua [Department of Oncology and Diagnostic Sciences, University of Maryland Dental School, 650W. Baltimore Street, 7-North, Baltimore, MD 21201 (United States); Binmadi, Nada O. [Department of Oncology and Diagnostic Sciences, University of Maryland Dental School, 650W. Baltimore Street, 7-North, Baltimore, MD 21201 (United States); Department of Oral Basic and Clinical Sciences, King Abdulaziz University, Jeddah 21589 (Saudi Arabia); Proia, Patrizia [Department of Oncology and Diagnostic Sciences, University of Maryland Dental School, 650W. Baltimore Street, 7-North, Baltimore, MD 21201 (United States); Department of Sports Science (DISMOT), University of Palermo, Via Eleonora Duse 2 90146, Palermo (Italy); Basile, John R., E-mail: jbasile@umaryland.edu [Department of Oncology and Diagnostic Sciences, University of Maryland Dental School, 650W. Baltimore Street, 7-North, Baltimore, MD 21201 (United States); Greenebaum Cancer Center, 22S. Greene Street, Baltimore, MD 21201 (United States)

    2012-08-15

    Growth and metastasis of solid tumors requires induction of angiogenesis to ensure the delivery of oxygen, nutrients and growth factors to rapidly dividing transformed cells. Through either mutations, hypoxia generated by cytoreductive therapies, or when a malignancy outgrows its blood supply, tumor cells undergo a change from an avascular to a neovascular phenotype, a transition mediated by the hypoxia-inducible factor (HIF) family of transcriptional regulators. Vascular endothelial growth factor (VEGF) is one example of a gene whose transcription is stimulated by HIF. VEGF plays a crucial role in promoting tumor growth and survival by stimulating new blood vessel growth in response to such stresses as chemotherapy or radiotherapy-induced hypoxia, and it therefore has become a tempting target for neutralizing antibodies in the treatment of advanced neoplasms. Emerging evidence has shown that the semaphorins, proteins originally associated with control of axonal growth and immunity, are regulated by changes in oxygen tension as well and may play a role in tumor-induced angiogenesis. Through the use of RNA interference, in vitro and in vivo angiogenesis assays and tumor xenograft experiments, we demonstrate that expression of semaphorin 4D (SEMA4D), which is under the control of the HIF-family of transcription factors, cooperates with VEGF to promote tumor growth and vascularity in oral squamous cell carcinoma (OSCC). We use blocking antibodies to show that targeting SEMA4D function along with VEGF could represent a novel anti-angiogenic therapeutic strategy for the treatment of OSCC and other solid tumors. -- Highlights: Black-Right-Pointing-Pointer Similar to VEGF, SEMA4D promotes angiogenesis in vitro and in vivo. Black-Right-Pointing-Pointer Both VEGF and SEMA4D are produced by OSCC cells in a HIF-dependent manner. Black-Right-Pointing-Pointer These factors combine to elicit a robust pro-angiogenic phenotype in OSCC. Black-Right-Pointing-Pointer Anti-SEMA4D

  1. In Vivo Hypoxia PET Imaging Quantifies the Severity of Arthritic Joint Inflammation in Line with Overexpression of Hypoxia-Inducible Factor and Enhanced Reactive Oxygen Species Generation.

    Science.gov (United States)

    Fuchs, Kerstin; Kuehn, Anna; Mahling, Moritz; Guenthoer, Philipp; Hector, Andreas; Schwenck, Johannes; Hartl, Dominik; Laufer, Stefan; Kohlhofer, Ursula; Quintanilla-Martinez, Leticia; Reischl, Gerald; Röcken, Martin; Pichler, Bernd J; Kneilling, Manfred

    2017-05-01

    Hypoxia is essential for the development of autoimmune diseases such as rheumatoid arthritis (RA) and is associated with the expression of reactive oxygen species (ROS), because of the enhanced infiltration of immune cells. The aim of this study was to demonstrate the feasibility of measuring hypoxia noninvasively in vivo in arthritic ankles with PET/MRI using the hypoxia tracers 18F-fluoromisonidazole (18F-FMISO) and 18F-fluoroazomycinarabinoside (18F-FAZA). Additionally, we quantified the temporal dynamics of hypoxia and ROS stress using L-012, an ROS-sensitive chemiluminescence optical imaging probe, and analyzed the expression of hypoxia-inducible factors (HIFs). Methods: Mice underwent noninvasive in vivo PET/MRI to measure hypoxia or optical imaging to analyze ROS expression. Additionally, we performed ex vivo pimonidazole-/HIF-1α immunohistochemistry and HIF-1α/2α Western blot/messenger RNA analysis of inflamed and healthy ankles to confirm our in vivo results. Results: Mice diseased from experimental RA exhibited a 3-fold enhancement in hypoxia tracer uptake, even in the early disease stages, and a 45-fold elevation in ROS expression in inflamed ankles compared with the ankles of healthy controls. We further found strong correlations of our noninvasive in vivo hypoxia PET data with pimonidazole and expression of HIF-1α in arthritic ankles. The strongest hypoxia tracer uptake was observed as soon as day 3, whereas the most pronounced ROS stress was evident on day 6 after the onset of experimental RA, indicating that tissue hypoxia can precede ROS stress in RA. Conclusion: Collectively, for the first time to our knowledge, we have demonstrated that the noninvasive measurement of hypoxia in inflammation using 18F-FAZA and 18F-FMISO PET imaging represents a promising new tool for uncovering and monitoring rheumatic inflammation in vivo. Further, because hypoxic inflamed tissues are associated with the overexpression of HIFs, specific inhibition of HIFs

  2. The 2-oxoglutarate analog 3-oxoglutarate decreases normoxic hypoxia-inducible factor-1α in cancer cells, induces cell death, and reduces tumor xenograft growth

    Directory of Open Access Journals (Sweden)

    Koivunen P

    2016-03-01

    Full Text Available Peppi Koivunen,1 Stuart M Fell,2,3 Wenyun Lu,4 Joshua D Rabinowitz,4 Andrew L Kung,5,6 Susanne Schlisio,2,7 1Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland; 2Ludwig Institute for Cancer Research Ltd, Stockholm, Sweden; 3Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden; 4Department of Chemistry and Integrative Genomics, Princeton University, Princeton, NJ, 5Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, 6Department of Pediatrics, Columbia University Medical Center, New York, NY, USA; 7Department of Microbiology and Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Abstract: The cellular response to hypoxia is primarily regulated by the hypoxia-inducible factors (HIFs. HIF-1α is also a major mediator of tumor physiology, and its abundance is correlated with therapeutic resistance in a broad range of cancers. Accumulation of HIF-1α under hypoxia is mainly controlled by the oxygen-sensing HIF prolyl 4-hydroxylases (EGLNs, also known as PHDs. Here, we identified a high level of normoxic HIF-1α protein in various cancer cell lines. EGLNs require oxygen and 2-oxoglutarate for enzymatic activity. We tested the ability of several cell-permeable 2-oxoglutarate analogs to regulate the abundance of HIF-1α protein. We identified 3-oxoglutarate as a potent regulator of HIF-1α in normoxic conditions. In contrast to 2-oxoglutarate, 3-oxoglutarate decreased the abundance of HIF-1α protein in several cancer cell lines in normoxia and diminished HIF-1α levels independent of EGLN enzymatic activity. Furthermore, we observed that 3-oxoglutarate was detrimental to cancer cell survival. We show that esterified 3-oxoglutarate, in combination with the cancer chemotherapeutic drug vincristine, induces apoptosis and inhibits tumor growth in vitro and in vivo. Our data

  3. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer

    Science.gov (United States)

    2013-10-01

    obese and overweight patients with ER+ breast cancer to neoadjuvant aromatase inhibitor therapy. My role in this clinical trial is to analyze HIF-1 and...with drug resistance in different cancer cell types, including chronic myeloid leukemia cells (Zhao et al. Oncogene. 2010), gastric cancer cells (Liu...Hypoxia- Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer PRINCIPAL INVESTIGATOR: Armina Kazi CONTRACTING

  4. Hypoxia-induced downregulation of autophagy mediator Beclin 1 reduces the susceptibility of malignant intestinal epithelial cells to hypoxia-dependent apoptosis.

    Science.gov (United States)

    Yoo, Byong Hoon; Wu, Xue; Derouet, Mathieu; Haniff, Mehnaaz; Eskelinen, Eeva-Liisa; Rosen, Kirill

    2009-11-01

    Disruption of tumor blood supply causes tumor hypoxia. Hypoxia can induce cell death, but cancer cells that remain viable in the absence of oxygen often possess an increased survival potential, and tumors formed by these cells tend to grow particularly aggressively. Thus, developing approaches aimed at increasing the susceptibility of malignant cells to hypoxia-induced death represents a potentially important avenue for cancer treatment. Molecular mechanisms that control the survival of cancer cells under hypoxia are not well understood. In an effort to understand them we found that hypoxia downregulates Beclin 1, a mediator of autophagy, in malignant intestinal epithelial cells. The reversal of this downregulation promoted autophagosome accumulation, enhanced the activation of a pro-apoptotic protease caspase-9 and subsequent caspase-9-dependent activation of two other pro-apoptotic proteases caspases 3 and 7 in these cells. Furthermore, the reversal of hypoxia-induced downregulation of Beclin 1-stimulated caspase-9-dependent apoptosis of the indicated cells under hypoxia. Interestingly, we found that Beclin 1-dependent caspase-9 activation in hypoxic cells was not associated with an increased release of cytochrome c from the mitochondria to the cytoplasm (such release represents a frequently occurring mechanism for caspase-9 activation). We also observed that Beclin 1-dependent apoptosis of hypoxic malignant cells was independent of FADD, a mediator of death receptor signaling. We conclude that hypoxia triggers a feedback mechanism that delays apoptosis of oxygen-deprived malignant intestinal epithelial cells and is driven by hypoxia-induced Beclin 1 downregulation. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of such cells to hypoxia-induced apoptosis.

  5. Effect of Moderate Hepatic Impairment on the Pharmacokinetics and Pharmacodynamics of Roxadustat, an Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor

    OpenAIRE

    Groenendaal-van de Meent, Dorien; Adel, Martin den; Noukens, Jan; Rijnders, Sanne; Krebs-Brown, Axel; Mateva, Lyudmila; Alexiev, Assen; Schaddelee, Marloes

    2016-01-01

    Background and Objective Roxadustat is a hypoxia-inducible factor prolyl hydroxylase inhibitor in phase III development for the treatment of anaemia associated with chronic kidney disease. This study evaluated the effects of moderate hepatic impairment on roxadustat pharmacokinetics, pharmacodynamics and tolerability. Methods This was an open-label study in which eight subjects with moderate hepatic impairment (liver cirrhosis Child?Pugh score 7?9) and eight subjects with normal hepatic funct...

  6. Senp1 drives hypoxia-induced polycythemia via GATA1 and Bcl-xL in subjects with Monge's disease.

    Science.gov (United States)

    Azad, Priti; Zhao, Huiwen W; Cabrales, Pedro J; Ronen, Roy; Zhou, Dan; Poulsen, Orit; Appenzeller, Otto; Hsiao, Yu Hsin; Bafna, Vineet; Haddad, Gabriel G

    2016-11-14

    In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, respectively, CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding, we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. © 2016 Azad et al.

  7. Changes in reactive oxygen species, superoxide dismutase, and hypoxia-inducible factor-1α levels in missed abortion.

    Science.gov (United States)

    Zhu, Li-Jun; Chen, Ya-Ping; Chen, Bing-Jin; Mei, Xiao-Hui

    2014-01-01

    This study aimed to investigate changes in the expression levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and hypoxia-inducible factor-1α (HIF-1α) in the trophoblasts of patients who had experienced missed abortions. The missed abortion group included 28 patients with missed abortions. The control group was comprised of 35 women who had elected to undergo surgically induced abortion in their first trimester, and whose embryos were confirmed to be alive before surgery. No woman in either group had any known causative factor for missed or spontaneous abortion. As soon as the diagnosis of "missed abortion" was definitively made, the chorionic trophoblast was obtained by induced abortion operation. The same method was used for individuals in the control group, who were at 7-10 weeks of pregnancy. Levels of ROS, SOD, and HIF-1α in the chorionic trophoblasts from women in both groups were examined within 1 hour by fluorescent staining, chemiluminometry, and enzyme immunoassay methods. The SOD and HIF-1α levels were lower and the ROS level was higher in the trophoblasts from women in the missed abortion group compared to levels in the control group (P abortion are altered compared to levels in control patients. Changes in these factors should be evaluated further for their potential role in missed abortion.

  8. Distinctive expression patterns of hypoxia-inducible factor-1α and endothelial nitric oxide synthase following hypergravity exposure.

    Science.gov (United States)

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-06-07

    This study was designed to examine the expression of hypoxia-inducible factor-1α (HIF-1α) and the level and activity of endothelial nitric oxide synthase (eNOS) in the hearts and livers of mice exposed to hypergravity. Hypergravity-induced hypoxia and the subsequent post-exposure reoxygenation significantly increased cardiac HIF-1α levels. Furthermore, the levels and activity of cardiac eNOS also showed significant increase immediately following hypergravity exposure and during the reoxygenation period. In contrast, the expression of phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) showed significant elevation only during the reoxygenation period. These data raise the possibility that the increase in cardiac HIF-1α expression induced by reoxygenation involves a cascade of signaling events, including activation of the Akt and ERK pathways. In the liver, HIF-1α expression was significantly increased immediately after hypergravity exposure, indicating that hypergravity exposure to causes hepatocellular hypoxia. The hypergravity-exposed livers showed significantly higher eNOS immunoreactivity than did those of control mice. Consistent with these results, significant increases in eNOS activity and nitrate/nitrite levels were also observed. These findings suggest that hypergravity-induced hypoxia plays a significant role in the upregulation of hepatic eNOS.

  9. 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.

  10. Hypoxia-induced HIF1α targets in melanocytes reveal a molecular profile associated with poor melanoma prognosis.

    Science.gov (United States)

    Loftus, Stacie K; Baxter, Laura L; Cronin, Julia C; Fufa, Temesgen D; Pavan, William J

    2017-05-01

    Hypoxia and HIF1α signaling direct tissue-specific gene responses regulating tumor progression, invasion, and metastasis. By integrating HIF1α knockdown and hypoxia-induced gene expression changes, this study identifies a melanocyte-specific, HIF1α-dependent/hypoxia-responsive gene expression signature. Integration of these gene expression changes with HIF1α ChIP-Seq analysis identifies 81 HIF1α direct target genes in melanocytes. The expression levels for 10 of the HIF1α direct targets - GAPDH, PKM, PPAT, DARS, DTWD1, SEH1L, ZNF292, RLF, AGTRAP, and GPC6 - are significantly correlated with reduced time of disease-free status in melanoma by logistic regression (P-value = 0.0013) and ROC curve analysis (AUC = 0.826, P-value < 0.0001). This HIF1α-regulated profile defines a melanocyte-specific response under hypoxia, and demonstrates the role of HIF1α as an invasive cell state gatekeeper in regulating cellular metabolism, chromatin and transcriptional regulation, vascularization, and invasion. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  11. Augmentation of leptin and hypoxia-inducible factor 1alpha mRNAs in the pre-eclamptic placenta.

    Science.gov (United States)

    Iwagaki, S; Yokoyama, Y; Tang, L; Takahashi, Y; Nakagawa, Y; Tamaya, T

    2004-05-01

    The placenta is a major source of leptin in the fetomaternal circulation, although its physiological role remains to be clarified. Leptin in the fetomaternal circulation is proposed to be a marker of acute stress in the fetus, and the fetus suffering from pre-eclampsia would be under chronic stress. In 16 pre-eclamptic placentas, the expressions of leptin, hypoxia-inducible factor 1alpha (HIF1alpha) and leptin receptor mRNAs were analyzed by semi-quantitative reverse-transcriptase-polymerase chain reaction and compared with clinical data. The co-expressions of leptin and the isoforms of the leptin receptor were observed in all the pre-eclamptic placentas. Leptin mRNA was significantly augmented in the pre-eclamptic placentas, although the level in fetal plasma was not high. The level of the expression of leptin mRNA was correlated with the placental HIF1alpha mRNA level and fetal body weight, but not with the levels of the leptin receptor isoforms in the pre-eclamptic placentas. This observation may suggest that autocrine/paracrine regulation of leptin exists in the human placenta and is upregulated in the pre-eclamptic placenta.

  12. Hypoxia-inducible factor-1α activation improves renal oxygenation and mitochondrial function in early chronic kidney disease.

    Science.gov (United States)

    Thomas, Joanna L; Pham, Hai; Li, Ying; Hall, Elanore; Perkins, Guy A; Ali, Sameh S; Patel, Hemal H; Singh, Prabhleen

    2017-08-01

    The pathophysiology of chronic kidney disease (CKD) is driven by alterations in surviving nephrons to sustain renal function with ongoing nephron loss. Oxygen supply-demand mismatch, due to hemodynamic adaptations, with resultant hypoxia, plays an important role in the pathophysiology in early CKD. We sought to investigate the underlying mechanisms of this mismatch. We utilized the subtotal nephrectomy (STN) model of CKD to investigate the alterations in renal oxygenation linked to sodium (Na) transport and mitochondrial function in the surviving nephrons. Oxygen delivery was significantly reduced in STN kidneys because of lower renal blood flow. Fractional oxygen extraction was significantly higher in STN. Tubular Na reabsorption was significantly lower per mole of oxygen consumed in STN. We hypothesized that decreased mitochondrial bioenergetic capacity may account for this and uncovered significant mitochondrial dysfunction in the early STN kidney: higher oxidative metabolism without an attendant increase in ATP levels, elevated superoxide levels, and alterations in mitochondrial morphology. We further investigated the effect of activation of hypoxia-inducible factor-1α (HIF-1α), a master regulator of cellular hypoxia response. We observed significant improvement in renal blood flow, glomerular filtration rate, and tubular Na reabsorption per mole of oxygen consumed with HIF-1α activation. Importantly, HIF-1α activation significantly lowered mitochondrial oxygen consumption and superoxide production and increased mitochondrial volume density. In conclusion, we report significant impairment of renal oxygenation and mitochondrial function at the early stages of CKD and demonstrate the beneficial role of HIF-1α activation on renal function and metabolism.

  13. Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.

    Science.gov (United States)

    Partch, Carrie L; Gardner, Kevin H

    2011-05-10

    Hypoxia-inducible factor (HIF) is the key transcriptional effector of the hypoxia response in eukaryotes, coordinating the expression of genes involved in oxygen transport, glycolysis, and angiogenesis to promote adaptation to low oxygen levels. HIF is a basic helix-loop-helix (bHLH)-PAS (PER-ARNT-SIM) heterodimer composed of an oxygen-labile HIF-α subunit and a constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) subunit, which dimerize via basic helix-loop-helix and PAS domains, and recruit coactivators via HIF-α C-terminal transactivation domains. Here we demonstrate that the ARNT PAS-B domain provides an additional recruitment site by binding the coactivator transforming acidic coiled-coil 3 (TACC3) in a step necessary for transcriptional responses to hypoxia. Structural insights from NMR spectroscopy illustrate how this PAS domain simultaneously mediates interactions with HIF-α and TACC3. Finally, mutations on ARNT PAS-B modulate coactivator selectivity and target gene induction by HIF in vivo, demonstrating a bifunctional role for transcriptional regulation by PAS domains within bHLH-PAS transcription factors.

  14. Role of Hypoxia Inducible Factor-1α (HIF-1α) in Innate Defense against Uropathogenic Escherichia coli Infection

    Science.gov (United States)

    Lin, Ann E.; Beasley, Federico C.; Olson, Joshua; Keller, Nadia; Shalwitz, Robert A.; Hannan, Thomas J.; Hultgren, Scott J.; Nizet, Victor

    2015-01-01

    Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infections (UTI) affecting approximately 150 million people worldwide. Here, we revealed the importance of transcriptional regulator hypoxia-inducible factor-1 α subunit (HIF-1α) in innate defense against UPEC-mediated UTI. The effects of AKB-4924, a HIF-1α stabilizing agent, were studied using human uroepithelial cells (5637) and a murine UTI model. UPEC adherence and invasion were significantly reduced in 5637 cells when HIF-1α protein was allowed to accumulate. Uroepithelial cells treated with AKB-4924 also experienced reduced cell death and exfoliation upon UPEC challenge. In vivo, fewer UPEC were recovered from the urine, bladders and kidneys of mice treated transurethrally with AKB-4924, whereas increased bacteria were recovered from bladders of mice with a HIF-1α deletion. Bladders and kidneys of AKB-4924 treated mice developed less inflammation as evidenced by decreased pro-inflammatory cytokine release and neutrophil activity. AKB-4924 impairs infection in uroepithelial cells and bladders, and could be correlated with enhanced production of nitric oxide and antimicrobial peptides cathelicidin and β-defensin-2. We conclude that HIF-1α transcriptional regulation plays a key role in defense of the urinary tract against UPEC infection, and that pharmacological HIF-1α boosting could be explored further as an adjunctive therapy strategy for serious or recurrent UTI. PMID:25927232

  15. Transient ureteral obstruction prevents against kidney ischemia/reperfusion injury via hypoxia-inducible factor (HIF-2α activation.

    Directory of Open Access Journals (Sweden)

    Shun Zhang

    Full Text Available Although the protective effect of transient ureteral obstruction (UO prior to ischemia on subsequent renal ischemia/reperfusion (I/R injury has been documented, the underlying molecular mechanism remains to be understood. We showed in the current study that 24 h of UO led to renal tubular hypoxia in the ipsilateral kidney in mice, with the accumulation of hypoxia-inducible factor (HIF-2α, which lasted for a week after the release of UO. To address the functions of HIF-2α in UO-mediated protection of renal IRI, we utilized the Mx-Cre/loxP recombination system to knock out target genes. Inactivation of HIF-2α, but not HIF-1α blunted the renal protective effects of UO, as demonstrated by much higher serum creatinine level and severer histological damage. UO failed to prevent postischemic neutrophil infiltration and apoptosis induction in HIF-2α knockout mice, which also diminished the postobstructive up-regulation of the protective molecule, heat shock protein (HSP-27. The renal protective effects of UO were associated with the improvement of the postischemic recovery of intra-renal microvascular blood flow, which was also dependent on the activation of HIF-2α. Our results demonstrated that UO protected the kidney via activation of HIF-2α, which reduced tubular damages via preservation of adequate renal microvascular perfusion after ischemia. Thus, preconditional HIF-2α activation might serve as a novel therapeutic strategy for the treatment of ischemic acute renal failure.

  16. Transcriptional repression of Na-K-2Cl cotransporter NKCC1 by hypoxia-inducible factor-1.

    Science.gov (United States)

    Ibla, Juan C; Khoury, Joseph; Kong, Tianqing; Robinson, Andreas; Colgan, Sean P

    2006-08-01

    Tissue edema is commonly associated with hypoxia. Generally, such episodes of fluid accumulation are self-limiting. At present, little is known about mechanisms to compensate excessive fluid transport. Here we describe an adaptive mechanism to dampen fluid loss during hypoxia. Initial studies confirmed previous observations of attenuated electrogenic Cl- secretion after epithelial hypoxia. A screen of known ion transporters in Cl- -secreting epithelia revealed selective downregulation of Na-K-2Cl cotransporter NKCC1 mRNA, protein, and function. Subsequent studies identified transcriptional repression of NKCC1 mediated by hypoxia-inducible factor (HIF). Chromatin immunoprecipitation analysis identified a functional HIF binding site oriented on the antisense strand of genomic DNA downstream of the transcription start site corresponding to the NKCC1 5'-untranslated region. Additional in vivo studies using conditional Hif1a-null mice revealed that the loss of HIF-1alpha in Cl- -secreting epithelia results in a loss of NKCC1 repression. These studies describe a novel regulatory pathway for NKCC1 transcriptional repression by hypoxia. These results suggest that HIF-dependent repression of epithelial NKCC1 may provide a compensatory mechanism to prevent excessive fluid loss during hypoxia.

  17. Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors: A Potential New Treatment for Anemia in Patients With CKD.

    Science.gov (United States)

    Gupta, Nupur; Wish, Jay B

    2017-06-01

    Erythropoiesis-stimulating agents (ESAs) increase hemoglobin levels, reduce transfusion requirements, and have been the standard of treatment for anemia in patients with chronic kidney disease (CKD) since 1989. Many safety concerns have emerged regarding the use of ESAs, including an increased occurrence of cardiovascular events and vascular access thrombosis. Hypoxia-inducible factor (HIF) prolyl hydroxylase (PH) enzyme inhibitors are a new class of agents for the treatment of anemia in CKD. These agents work by stabilizing the HIF complex and stimulating endogenous erythropoietin production even in patients with end-stage kidney disease. HIF-PH inhibitors improve iron mobilization to the bone marrow. They are administered orally, which may be a more favorable route for patients not undergoing hemodialysis. By inducing considerably lower but more consistent blood erythropoietin levels than ESAs, HIF-PH inhibitors may be associated with fewer adverse cardiovascular effects at comparable hemoglobin levels, although this has yet to be proved in long-term clinical trials. One significant concern regarding the long-term use of these agents is their possible effect on tumor growth. There are 4 such agents undergoing phase 2 and 3 clinical trials in the United States; this report provides a focused review of HIF-PH inhibitors and their potential clinical utility in the management of anemia of CKD. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. 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.

  19. Hypoxia-inducible factor 1α predicts recurrence in high-grade soft tissue sarcoma of extremities and trunk wall

    DEFF Research Database (Denmark)

    Nyström, Harriet; Jönsson, M; Werner-Hartman, L

    2017-01-01

    BACKGROUND AND AIM: Sarcomas are of mesenchymal origin and typically show abundant tumour stroma and presence of necrosis. In search for novel biomarkers for personalised therapy, we determined the prognostic impact of stromal markers, hypoxia and neovascularity in high-grade soft tissue leiomyos......BACKGROUND AND AIM: Sarcomas are of mesenchymal origin and typically show abundant tumour stroma and presence of necrosis. In search for novel biomarkers for personalised therapy, we determined the prognostic impact of stromal markers, hypoxia and neovascularity in high-grade soft tissue...... leiomyosarcoma and pleomorphic undifferentiated sarcoma. METHOD: We evaluated CD163, colony-stimulating factor (CSF)-1, CD16 and hypoxia-inducible factor 1 (HIF-1)α using immunohistochemical staining and assessed microvessel density using CD31 in 73 high-grade leiomyosarcomas and undifferentiated pleomorphic...... sarcomas of the extremities and the trunk wall. The results were correlated to metastasis-free and overall survival. RESULTS: Expression of HIF-1α was associated with the presence of necrosis and independently predicted shorter metastasis-free survival (HR 3.2, CI 1.4 to 7.0, p=0.004), whereas neither...

  20. Molecular cloning and characterization of the Xenopus hypoxia-inducible factor 1alpha (xHIF1alpha).

    Science.gov (United States)

    de Beaucourt, Arnaud; Coumailleau, Pascal

    2007-12-15

    We report the molecular cloning and the characterization of the Xenopus homolog of mammalian hypoxia-inducible factor 1alpha (HIF1alpha), a member of the bHLH/PAS transcription factor family. Searches in Xenopus genome sequences and phylogenetic analysis reveal the existence of HIF1alpha and HIF2alpha paralogs in the Xenopus laevis species. Sequence data analyses indicate that the organization of protein domains in Xenopus HIF1alpha (xHIF1alpha) is strongly conserved. We also show that xHIF1alpha heterodimerizes with the Xenopus Arnt1 protein (xArnt1) with the proteic complex being mediated by the HLH and PAS domains. Subcellular analysis in a Xenopus XTC cell line using chimeric GFP constructs show that over-expression of xHIF1alpha and xArnt1 allows us to detect the xHIF1alpha/xArnt1 complex in the nucleus, but only in the presence of both partners. Further analyses in XTC cell line show that over-producing xHIF1alpha and xArnt1 mediates trans-activation of the hypoxia response element (HRE) reporter. The trans-activation level can be increased in hypoxia conditions. Interestingly such trans-activation properties can be also observed when human Arnt1 is used together with the xHIF1alpha. Copyright (c) 2007 Wiley-Liss, Inc.

  1. Expression of hypoxia-inducible factor-1α during ovarian follicular growth and development in Sprague-Dawley rats.

    Science.gov (United States)

    Zhang, Z H; Chen, L Y; Wang, F; Wu, Y Q; Su, J Q; Huang, X H; Wang, Z C; Cheng, Y

    2015-06-01

    Hypoxia-inducible factor-1α (HIF-1α) has been identified as a transcription factor that is involved in diverse physiological and pathological processes in the ovary. In this study, we examined whether HIF-1α is expressed in a cell- and stage-specific manner during follicular growth and development in the mammalian ovaries. Using immunohistochemistry and Western blot analysis, HIF-1α expression was observed in granulosa cells specifically and was significantly increased during the follicular growth and development of postnatal rats. Furthermore, pregnant mare serum gonadotropin also induced HIF-1α expression in granulosa cells and ovaries during the follicular development of immature rats primed with gonadotropin. Moreover, we also examined proliferation cell nuclear antigen, a cell proliferation marker, during follicular growth and development and found that its expression pattern was similar to that of HIF-1α protein. Granulosa cell culture experiments revealed that proliferation cell nuclear antigen expression may be regulated by HIF-1α. These results indicated that HIF-1α plays an important role in the follicular growth and development of these 2 rat models. The HIF-1α-mediated signaling pathway may be an important mechanism regulating follicular growth and development in mammalian ovaries in vivo.

  2. Hypoxia-inducible factor-mediated induction of WISP-2 contributes to attenuated progression of breast cancer.

    Science.gov (United States)

    Fuady, Jerry H; Bordoli, Mattia R; Abreu-Rodríguez, Irene; Kristiansen, Glen; Hoogewijs, David; Stiehl, Daniel P; Wenger, Roland H

    2014-01-01

    Hypoxia and the hypoxia-inducible factor (HIF) signaling pathway trigger the expression of several genes involved in cancer progression and resistance to therapy. Transcriptionally active HIF-1 and HIF-2 regulate overlapping sets of target genes, and only few HIF-2 specific target genes are known so far. Here we investigated oxygen-regulated expression of Wnt-1 induced signaling protein 2 (WISP-2), which has been reported to attenuate the progression of breast cancer. WISP-2 was hypoxically induced in low-invasive luminal-like breast cancer cell lines at both the messenger RNA and protein levels, mainly in a HIF-2α-dependent manner. HIF-2-driven regulation of the WISP2 promoter in breast cancer cells is almost entirely mediated by two phylogenetically and only partially conserved functional hypoxia response elements located in a microsatellite region upstream of the transcriptional start site. High WISP-2 tumor levels were associated with increased HIF-2α, decreased tumor macrophage density, and a better prognosis. Silencing WISP-2 increased anchorage-independent colony formation and recovery from scratches in confluent cell layers of normally low-invasive MCF-7 cancer cells. Interestingly, these changes in cancer cell aggressiveness could be phenocopied by HIF-2α silencing, suggesting that direct HIF-2-mediated transcriptional induction of WISP-2 gene expression might at least partially explain the association of high HIF-2α tumor levels with prolonged overall survival of patients with breast cancer.

  3. Antenatal hypoxia induces epigenetic repression of glucocorticoid receptor and promotes ischemic-sensitive phenotype in the developing heart.

    Science.gov (United States)

    Xiong, Fuxia; Lin, Thant; Song, Minwoo; Ma, Qingyi; Martinez, Shannalee R; Lv, Juanxiu; MataGreenwood, Eugenia; Xiao, Daliao; Xu, Zhice; Zhang, Lubo

    2016-02-01

    Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at -4408 and -3896 and Sp1 binding sites at -3425 and -3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2'-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Cross-talk between constitutive androstane receptor and hypoxia-inducible factor in the regulation of gene expression.

    Science.gov (United States)

    Shizu, Ryota; Shindo, Sawako; Yoshida, Takemi; Numazawa, Satoshi

    2013-05-23

    Hypoxia inducible factor (HIF) and 5'-AMP-activated protein kinase are often activated under similar physiological conditions. Constitutive androstane receptor (CAR) translocates into the nucleus in accordance with 5'-AMP-activated protein kinase and thus confers transactivation. The aim of the present study was to investigate a possible link between CAR and HIFα. Phenobarbital (PB), a typical CAR activator, increased the gene expression of HIF-target genes in the livers of mice, including erythropoietin, heme oxygenase-1 and vascular endothelial growth factor-a. PB induced an accumulation of nuclear HIF-1α and an increase in the HIF-responsive element-mediated transactivation in HepG2 cells. Cobalt chloride, a typical HIF activator, induced the gene expression of CAR-target genes, including cyp2b9 and cyp2b10, an accumulation of nuclear CAR and an increase in the PB-responsive enhancer module-mediated transactivation in the mouse liver. Immunoprecipitation-immunoblot and chromatin immunoprecipitation analyses suggest that CAR binds to the PB-responsive enhancer module with HIF-1α in the liver of untreated mice and that the complex dissociates upon PB treatment. Taken together these results suggest that CAR and HIF-α interact and reciprocally modulate the functions of each other. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Chronic hypoxia-inducible transcription factor-2 activation stably transforms juxtaglomerular renin cells into fibroblast-like cells in vivo.

    Science.gov (United States)

    Kurt, Birguel; Gerl, Katharina; Karger, Christian; Schwarzensteiner, Ilona; Kurtz, Armin

    2015-03-01

    On the basis of previous observations that deletion of the von Hippel-Lindau protein (pVHL) in juxtaglomerular (JG) cells of the kidney suppresses renin and induces erythropoietin expression, this study aimed to characterize the events underlying this striking change of hormone expression. We found that renin cell-specific deletion of pVHL in mice leads to a phenotype switch in JG cells, from a cuboid and multiple vesicle-containing form into a flat and elongated form without vesicles. This shift of cell phenotype was accompanied by the disappearance of marker proteins for renin cells (e.g., aldo-keto reductase family 1, member 7 and connexin 40) and by the appearance of markers of fibroblast-like cells (e.g., collagen I, ecto-5'-nucleotidase, and PDGF receptor-β). Furthermore, hypoxia-inducible transcription factor-2α (HIF-2α) protein constitutively accumulated in these transformed cells. Codeletion of pVHL and HIF-2α in JG cells completely prevented the phenotypic changes. Similar to renin expression in normal JG cells, angiotensin II negatively regulated erythropoietin expression in the transformed cells. In summary, chronic activation of HIF-2 in renal JG cells leads to a reprogramming of the cells into fibroblast-like cells resembling native erythropoietin-producing cells located in the tubulointerstitium. Copyright © 2015 by the American Society of Nephrology.

  6. Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease.

    Science.gov (United States)

    Das, Nupur; Xie, Liwei; Ramakrishnan, Sadeesh K; Campbell, Andrew; Rivella, Stefano; Shah, Yatrik M

    2015-09-25

    Sickle cell disease (SCD) is caused by genetic defects in the β-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation

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    Stephan Niebler

    2015-01-01

    Full Text Available The transcription factor AP-2ε (activating enhancer-binding protein epsilon is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4 strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1, the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2′-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  8. Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

    Science.gov (United States)

    Chang, Wei-Lun; Chang, Yi-Cheng; Lin, Kuan-Ting; Li, Han-Ru; Pai, Chih-Yu; Chen, Jen-Hao; Su, Yi-Hsien

    2017-08-15

    Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus , hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development. © 2017. Published by The Company of Biologists Ltd.

  9. Effect of Hachimijiogan against Renal Dysfunction and Involvement of Hypoxia-Inducible Factor-1α in the Remnant Kidney Model

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    Hiroshi Oka

    2011-01-01

    Full Text Available In chronic renal failure, hypoxia of renal tissue is thought to be the common final pathway leading to end-stage renal failure. In this study the effects of hachimijiogan, a Kampo formula, were studied with respect to hypoxia-inducible factor (HIF. Using remnant kidney rats, we studied the effects of hachimijiogan on renal function in comparison with angiotensin II receptor blocker. The result showed that oral administration of hachimijiogan for seven days suppressed urinary protein excretion and urinary 8-OHdG, a marker of antioxidant activity, equally as well as oral administration of candesartan cilexetil. In contrast, the protein volume of HIF-1α in the renal cortex was not increased in the candesartan cilexetil group, but that in the hachimijiogan group was increased. In immunohistochemical studies as well, the expression of HIF-1α of the high-dose hachimijiogan group increased compared to that of the control group. Vascular endothelial growth factor and glucose transporter 1, target genes of HIF-1α, were also increased in the hachimijiogan group. These results suggest that hachimijiogan produces a protective effect by a mechanism different from that of candesartan cilexetil.

  10. Decay accelerating factor (CD55 protects neuronal cells from chemical hypoxia-induced injury

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    Tsokos George C

    2010-04-01

    Full Text Available Abstract Background Activated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the complement activation cascade may represent a potential therapeutic strategy for the management of ischemic brain injury. Decay-accelerating factor (DAF, also known as CD55 inhibits complement activation by suppressing the function of C3/C5 convertases, thereby limiting local generation or deposition of C3a/C5a and membrane attack complex (MAC or C5b-9 production. The present study investigates the ability of DAF to protect primary cultured neuronal cells subjected to sodium cyanide (NaCN-induced hypoxia from degeneration and apoptosis. Methods Cultured primary cortical neurons from embryonic Sprague-Dawley rats were assigned one of four groups: control, DAF treatment alone, hypoxic, or hypoxic treated with DAF. Hypoxic cultures were exposed to NaCN for 1 hour, rinsed, followed by 24 hour exposure to 200 ng/ml of recombinant human DAF in normal medium. Human DAF was used in the present study and it has been shown to effectively regulate complement activation in rats. Neuronal cell function, morphology and viability were investigated by measuring plateau depolarization potential, counting the number dendritic spines, and observing TUNEL and MTT assays. Complement C3, C3a, C3a receptor (R production, C3a-C3aR interaction and MAC formation were assessed along with the generation of activated caspase-9, activated caspase-3, and activated Src. Results When compared to controls, hypoxic cells had fewer dendritic spines, reduced plateau depolarization accompanied by increased apoptotic activity and accumulation of MAC, as well as up-regulation of C3, C3a and C3aR, enhancement of C3a-C3aR engagement, and elevated caspase and Src activity. Treatment of hypoxic cells with 200 ng/ml of recombinant human DAF resulted in attenuation of neuronal apoptosis and exerted

  11. Decreased Cyr61 under hypoxia induces extravillous trophoblasts apoptosis and preeclampsia.

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    Chen, Xi; Liu, Yanyan; Xu, Xiaoyan; Chen, Hanping

    2011-04-01

    During placental development, oxygen environment is not only critical for trophoblasts migration and invasion, but also fundamental for appropriate placental perfusion. Cysteine-rich 61 (Cyr61, CCN1) was expressed in the extravillous trophoblasts (EVTs) and decreased in preeclampsia. Its regulatory properties in human first-trimester extravillous trophoblast cell line (TEV-1 cells) upon a low oxygen tension were investigated. The present study examined functional changes involved in adaptation to hypoxia of the TEV-1 cells, using cobalt chloride (CoCl(2)) as hypoxic mimic. It was found that hypoxia inhibited growth of TEV-1 cells and induced the increase of cell apoptosis (Papoptosis has been implicated in the failure of trophoblasts to fully invade and modify the uterine environment and Cyr61 down-regulation, potentially leading to preeclampsia.

  12. Two mTOR inhibitors, rapamy