Radiosensitization of hypoxic tumor cells in vitro by nitric oxide

International Nuclear Information System (INIS)

Griffin, Robert J.; Makepeace, Carol M.; Hur, Won-Joo; Song, Chang W.

1996-01-01

Purpose: The effects of nitric oxide (NO) on the radiosensitivity of SCK tumor cells in oxic and hypoxic environments in vitro were studied. Methods and Materials: NO was delivered to cell suspensions using the NO donors 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), and a spermine/nitric oxide complex (SPER/NO), which release NO at half-lives of 2.1 min and 39 min at pH 7.4, respectively. The cells were suspended in media containing DEA/NO or SPER/NO for varying lengths of time under oxic or hypoxic conditions, irradiated, and the clonogenicity determined. Results: Both compounds markedly radiosensitized the hypoxic cells. The drug enhancement ratios (DER) for 0.1, 1.0, and 2.0 mM DEA/NO were 2.0, 2.3 and 3.0, respectively, and those for 0.1, 1.0, and 2.0 mM SPER/NO were 1.6, 2.3, and 2.8, respectively. Aerobic cells were not radiosensitized by DEA/NO or SPER/NO. When DEA/NO and SPER/NO were incubated in solution overnight to allow release of NO, they were found to have no radiosensitizing effect under hypoxic or oxic conditions indicating the sensitization by the NO donors was due to the NO molecule released from these drugs. At the higher concentrations, SPER/NO was found to be cytotoxic in aerobic conditions but not in hypoxic conditions. DEA/NO was only slightly toxic to the cells in both aerobic and hypoxic conditions. Conclusions: NO released from NO donors DEA/NO and SPER/NO is as effective as oxygen to radiosensitize hypoxic cells in vitro. Its application to the radiosensitization of hypoxic cells in solid tumors remains to be investigated

Cytotoxic properties of a 4-nitroimidazole (NSC 38087): a radiosensitizer of hypoxic cells in vitro

International Nuclear Information System (INIS)

Stratford, I.J.; Williamson, C.; Hardy, C.

1981-01-01

5-Phenoxysulphonyl-1-methyl-4-nitroimidazole (NSC 38087) can act as a sensitizer of hypoxic mammalian cells to radiation in vitro. The degree of sensitization achieved is greater than would be predicted from the drug's electron affinity. Cytotoxicity studies have shown that 5sub(μM) NSC 38087 can reduce the surviving fraction of log-phase V79 cells in air at 37 0 C to 10 -2 after 2 h exposure. This toxicity is considerably increased by small rises in temperature. In contrast to other nitroheterocyclic radiosensitizers, NSC 38087 and related 5-substituted 4-nitroimidazoles show greater toxicity towards aerobic than to hypoxic cells. Log-phase cells show the highest sensitivity to the toxic action of NSC 38087, with plateau-phase cells, cells with a history of chronic hypoxia, and thermotolerant cells showing greater resistance. These toxicity data are compared to those for the 2-nitroimidazole hypoxic-cell sensitizer misonidazole. (author)

Radiosensitization effect of CMNa on hypoxic pancreatic cancer cell in vitro

International Nuclear Information System (INIS)

Yin Lijie; Zhang Li; Ding Tiangui; Peng Zhaoxiang; Yu Huan; Gao Yuwei

2006-01-01

Objective: To investigate the effects of glycodidazolum natrium (CMNa) on pancreatic cancer cells under hypoxic condition. Methods: The human pancreatic cancer Panc-1 cells were exposed to a single fraction of high-dose γ-ray radiation either with CMNa or under hypoxic condition. The percentage of dead cells was detected with a multiwell plated reader, and fluorescence intensities of propidium iodide were measured before and after digitonin treatment. The sensitizing effect of CMNa on cell killing induced by high-dose irradiation was evaluated by time and concentration dependence. The selective radiosensitive effect of CMNa on hypoxia was evaluated by flow cytometry. Results: The death rate of pancreatic cancer Panc-1 cells paralleled with the increasing concentration of CMNa under hypoxic condition after 30 gray irradiation. The selective radiosensitive effect of CMNa on hypoxia was time-dependent. Conclusions: CMNa can enhance the radiosensitivity of pancreatic cancer Pane-1 cells under hypoxic condition with high-dose irradiation. (authors)

Turnover rate of hypoxic cells in solid tumors

International Nuclear Information System (INIS)

Ljungkvist, A.S.E.; Bussink, J.; Rijken, P.F.J.W.; Van Der Kogel, A.J.

2003-01-01

Most solid tumors contain hypoxic cells, and both the amount and duration of tumor hypoxia has been shown to influence the effect of radiation treatment negatively. It is important to understand the dynamic processes within the hypoxic cell population in non-treated tumors, and the effect of different treatment modalities on the kinetics of hypoxic cells to be able to design optimal combined modality treatments. The turnover rate of hypoxic cells was analyzed in three different solid tumor models with a double bio-reductive hypoxic marker assay with sequential injection of the two hypoxic markers. Previously it was shown that this assay could be used to detect both a decrease and an increase of tumor hypoxia in relation to the tumor vasculature with high spatial resolution. In this study the first hypoxic marker, pimonidazole, was administered at variable times relative to tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. The hypoxic cell turnover rate was calculated as the loss of pimonidazole positive cells relative to CCI-103F. The murine C38 line had the fastest hypoxic turnover rate of 60% /24h and the human xenograft line SCCNij3 had the slowest hypoxic turnover rate of 30% /24 h. The hypoxic turnover rate was most heterogeneous in the SCCNij3 line that even contained viable groups of cells that had been hypoxic for at least 5 days. The human xenograft line MEC82 fell in between with a hypoxic turnover rate of 50% /24 h. The hypoxic cell turnover was related to the potential tumor volume doubling time (Tpot) with a Tpot of 26h in C38 and 103h in SCCNij3. The dynamics of hypoxic cells, quantified with a double hypoxic marker method, showed large differences in hypoxic cell turnover rate and were related to Tpot

Radiosensitization conferred by oxygen and hypoxic cell sensitizers on human cells cultivated in vitro

International Nuclear Information System (INIS)

Pettersen, E.O.

1978-01-01

The main purpose was to provide additional information on two questions; (1) How does the radiosensitising effect of oxygen depend on oxygen concentration and cellular age, and (2) How does the radiosensitising effect of hypoxic cell sensitisers depend on concentration of sensitiser and cellular age. The general conclusions reached were as follows. The radiosensitising effect of oxygen on NHIK 3025 cells in G1 increased with increasing dose of radiation. For cells irradiated in S oxygen acted as a dose-modifying agent. For small doses of radiation the sensitising effect of oxygen was weaker for cells irradiated in G1 than for cells irradiated in S. The capacity of NHIK 3025 cells to repair sublethal damage after irradiation under extremely hypoxic conditions was low or even lost (even though the cells were subsequently incubated under aerobic conditions). The radiosensitising effect conferred by TMPN, diamide and misonidazole on NHIK 3025 cells was higher at high doses of radiation than at small doses of radiation (except for the dose-modifying radiosensitisation of cells in S by misonidazole). This observation supports arguments for using high dose fractions in fractionated radiotherapy where such chemicals are involved. (JIW)

Hypoxic radiosensitization: adored and ignored

DEFF Research Database (Denmark)

Overgaard, Jens

2007-01-01

resistance can be eliminated or modified by normobaric or hyperbaric oxygen or by the use of nitroimidazoles as hypoxic radiation sensitizers. More recently, attention has been given to hypoxic cytotoxins, a group of drugs that selectively or preferably destroys cells in a hypoxic environment. An updated......Since observations from the beginning of the last century, it has become well established that solid tumors may contain oxygen-deficient hypoxic areas and that cells in such areas may cause tumors to become radioresistant. Identifying hypoxic cells in human tumors has improved by the help of new...

The effect of dexamethasone on the radiation survival response and misonidazole-induced hypoxic-cell cytotoxicity in Chinese hamster cells V-79-753B in vitro

International Nuclear Information System (INIS)

Millar, B.C.; Jinks, S.

1981-01-01

Overnight exposure of Chinese hamster cells, V-79-753B, to microgram quantities of the synthetic corticosteroid, dexamethasone, resulted in a decrease in sensitivity towards radiation, both in air and in hypoxia. The effect was dose-modifying and the oxygen enhancement ratio did not change appreciably. Similarly, when dexamethasone-treated hypoxic cells were irradiated in the presence of misonidazole, a hypoxic cell radiosensitizer, there was a decrease in radiation sensitivity compared with untreated hypoxic cells irradiated with misonidazole. (author)

Hypoxic cell turnover in different solid tumor lines

International Nuclear Information System (INIS)

Ljungkvist, Anna S.E.; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

2005-01-01

Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h

Effects of bleomycin and irradiation on euoxic and hypoxic cells

International Nuclear Information System (INIS)

Shrieve, D.C.; Harris, J.W.

1979-01-01

EMT6 cells in vitro were exposed to bleomycin (BLM), either alone (under euoxic or hypoxic conditions) or in conjunction with x-radiation. Hypoxic and euoxic cells were equally sensitive to the drug in both of the systems used to induce hypoxia (ampules or chambers). Exposure to BLM immediately before x-irradiation altered the shape of the radiation survival curve decreasing the D 0 by a factor of 1.3. Simultaneous exposure to x-ray and BLM resulted in lower survivals than when radiation was given either before or after drug treatment. Cells recovered quickly from BLM damage if trypsinization was delayed. The results indicate that BLM and x-rays interact to lower cell survival but that cells recover from this effect if trypsinization is delayed

Experimental investigations on the relationship between radiation dose and sensitization of hypoxic cells by electron affinic compounds. Coordinated programme on improvement in radiotherapy of cancer using modifiers of radiosensitivity of cells

International Nuclear Information System (INIS)

Revesz, L.

1981-12-01

The investigations concern experimental studies on the factors which determine the inherent radiation response of mammalian cells, and the mechanism by which treatment with radiation protectors and hypoxic sensitizers modifies the response. Several mammalian cell lines including some derived from humans, were used in the tests of the biological response to radiation. Especially, the establishment of glutathione-deficient cell lines opened new experimental approaches to the question on the role of aminothiols in determining cellular radiation response. As the endpoints for the effect of radiation, single-strand DNA breaks by means of the sucrose gradient centrifugation and the unwinding technique in weak alkali, and colony forming ability of the cells were chosen. Radical reactions were also studied by the pulse-radiolysis technique. The enhancement of cellular radiosensitivity by oxygen and hypoxic cell sensitizers was found to be directly related to the glutathione level in the cells. Some particular aminothiols could substitute for the effect of glutathione in protecting against sensitization by oxygen and oxygen mimic sensitizers. The post irradiation repair of some DNA lesions induced by oxygen or hypoxic cell sensitizers was also associated with the level of glutathione and some specific aminothiols in the cells. The experiments revealed an efficient cellular cooperation in the repair of radiation induced DNA damage. Pulse radiolysis studies showed radical reactions characteristic for glutathione and not shared by other naturally occurring aminothiols. Inherent glutathione appears to play an important role in determining the intrinsic radiosensitivity of cells and the result of treatment with radioprotective and radiosensitizing substances. In particular, glutathione participates in both immediate radical reactions following exposure to ionizing radiation, and in the subsequent biochemical processes, and functions in promoting repair of the radiation damage

Priming of the Cells: Hypoxic Preconditioning for Stem Cell Therapy.

Science.gov (United States)

Wei, Zheng Z; Zhu, Yan-Bing; Zhang, James Y; McCrary, Myles R; Wang, Song; Zhang, Yong-Bo; Yu, Shan-Ping; Wei, Ling

2017-10-05

Stem cell-based therapies are promising in regenerative medicine for protecting and repairing damaged brain tissues after injury or in the context of chronic diseases. Hypoxia can induce physiological and pathological responses. A hypoxic insult might act as a double-edged sword, it induces cell death and brain damage, but on the other hand, sublethal hypoxia can trigger an adaptation response called hypoxic preconditioning or hypoxic tolerance that is of immense importance for the survival of cells and tissues. This review was based on articles published in PubMed databases up to August 16, 2017, with the following keywords: "stem cells," "hypoxic preconditioning," "ischemic preconditioning," and "cell transplantation." Original articles and critical reviews on the topics were selected. Hypoxic preconditioning has been investigated as a primary endogenous protective mechanism and possible treatment against ischemic injuries. Many cellular and molecular mechanisms underlying the protective effects of hypoxic preconditioning have been identified. In cell transplantation therapy, hypoxic pretreatment of stem cells and neural progenitors markedly increases the survival and regenerative capabilities of these cells in the host environment, leading to enhanced therapeutic effects in various disease models. Regenerative treatments can mobilize endogenous stem cells for neurogenesis and angiogenesis in the adult brain. Furthermore, transplantation of stem cells/neural progenitors achieves therapeutic benefits via cell replacement and/or increased trophic support. Combinatorial approaches of cell-based therapy with additional strategies such as neuroprotective protocols, anti-inflammatory treatment, and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the recent progress regarding cell types and applications in regenerative medicine as well as future applications.

The hypoxic tumor microenvironment and drug resistance against EGFR inhibitors: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines.

Science.gov (United States)

Boeckx, Carolien; Van den Bossche, Jolien; De Pauw, Ines; Peeters, Marc; Lardon, Filip; Baay, Marc; Wouters, An

2015-06-02

Increased expression of the epidermal growth factor receptor (EGFR) is observed in more than 90% of all head and neck squamous cell carcinomas (HNSCC). Therefore, EGFR has emerged as a promising therapeutic target. Nevertheless, drug resistance remains a major challenge and an important potential mechanism of drug resistance involves the hypoxic tumor microenvironment. Therefore, we investigated the cytotoxic effect of the EGFR-targeting agents cetuximab and erlotinib under normoxia versus hypoxia. Three cetuximab-sensitive HNSCC cell lines (SC263, LICR-HN2 and LICR-HN5) were treated with either cetuximab or erlotinib. Cells were incubated under normal or reduced oxygen conditions (<0.1% O2) for 24 or 72 h immediately after drug addition. Cell survival was assessed with the sulforhodamine B assay. Cetuximab and erlotinib established a dose-dependent growth inhibition under both normal and prolonged reduced oxygen conditions in all three HNSCC cell lines. However, a significantly increased sensitivity to cetuximab was observed in SC263 cells exposed to hypoxia for 72 h (p = 0.05), with IC50 values of 2.38 ± 0.59 nM, 0.64 ± 0.38 nM, and 0.10 ± 0.05 nM under normoxia, hypoxia for 24 h and hypoxia for 72 h, respectively. LICR-HN5 cells showed an increased sensitivity towards erlotinib when cells were incubated under hypoxia for 24 h (p = 0.05). Our results suggest that both EGFR-inhibitors cetuximab and erlotinib maintain their growth inhibitory effect under hypoxia. These results suggest that resistance to anti-EGFR therapy in HNSCC is probably not the result of hypoxic regions within the tumor and other mechanisms are involved.

Radiosensitizing and toxic effects of the 2-nitroimidazole Ro-07-0582 in different phases of the cell cycle of extremely hypoxic human cells in vitro

International Nuclear Information System (INIS)

Petterson, E.O.

1978-01-01

The radiosensitizing effect of 5 and 30 mM of Ro-07-0582 (misonidazole) has been studied at different stages of the cell cycle of mitotically selected NHIK 3025 cells under aerobic and extremely hypoxic conditions. For cells irradiated under aerobic conditions no sensitizing effect was seen at any stage of the cell cycle. For cells irradiated under extremely hypoxic conditions there was a lower sensitizing effect in mid-G1 than in mid-S for low radiation doses (in the initial region of the dose-response curves). For high radiation doses, however, no significant difference in sensitizing effect on cells in mid-G1 and in mid-S was seen. For cells in mid-G1 the sensitizing effect increased with increasing radiation dose. The toxic effect of 30 mM Ro-07-0582 as measured by loss of reproductive capacity was studied at room temperature for contact times up to 6 hours under aerobic conditions and 3 hours under extremely hypoxic conditions. While no effect was seen under aerobic conditions there was a toxic effect for contact intervals above 1 hour under extremely hypoxic conditions. Cells in S were more sensitive to the toxic effect of Ro-07-0582 than cells in G1. Implications for clinical use are discussed

Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

International Nuclear Information System (INIS)

Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

2016-01-01

The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba 2+ -sensitive inward rectifier K + current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca 2+ imaging study revealed that the hypoxic stress enhanced store-operated Ca 2+ (SOC) entry, which was significantly reduced in the presence of 100 μM Ba 2+ . On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba 2+ . We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca 2+ entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca 2+ (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia. �

Clusterin and chemotherapy sensitivity under normoxic and graded hypoxic conditions in colorectal cancer.

LENUS (Irish Health Repository)

Kevans, David

2012-06-01

In vitro studies have shown that clusterin modulates treatment sensitivity in a number of human cancers; however, the interaction between clusterin expression and hypoxia in controlling treatment response in CRC has not previously been examined. The aim of this study was to assess the effect of clusterin overexpression in CRC cells on sensitivity to 5-fluorouracil (5-FU), oxaliplatin and FOLFOX treatment under normoxic and graded hypoxic conditions.

Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

OpenAIRE

Harrison, Luke R.E.; Micha, Dimitra; Brandenburg, Martin; Simpson, Kathryn L.; Morrow, Christopher J.; Denneny, Olive; Hodgkinson, Cassandra; Yunus, Zaira; Dempsey, Clare; Roberts, Darren; Blackhall, Fiona; Makin, Guy; Dive, Caroline

2011-01-01

Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic indu...

Effect of electron affinic hypoxic cell sensitizers on the radiolytic depletion of oxygen in mammalian cells irradiated at ultrahigh dose rates

International Nuclear Information System (INIS)

Michaels, H.B.

1982-01-01

When CHO cells are equilibrated with a low level of oxygen (e.g. 0.4% O 2 ) and irradiated with single 3 ns pulses of electrons, a breaking survival curve is observed. This effect is believed to be the result of radiolytic oxygen depletion and can be prevented by the presence of a relatively low concentraton of hypoxic cell radiosensitizer. This prevention of the breaking survival curve has been observed for 2- and 5-nitroimidazoles, nitrofurans, and diamide. It is hypothesized that the sensitizer acts by competing wth oxygen for the radiation-induced intracellular oxygen-binding species, perhaps a hydrated electron adduct, leaving oxygen free to participate in radiosensitization reactions during the lifetime of the oxygen-sensitive radiation-induced target sites for lethal damage, probably DNA radicals produced by hydroxyl radical attack. The proposed role of the sensitizer in the interference with oxygen depletion is a transient phenomenon, occuring on the microsecond to millisecond time scale

Radiation-induced changes in nucleoid halo diameteres of aerobic and hypoxic SF-126 human brain tumor cells

International Nuclear Information System (INIS)

Wang, J.; Basu, H.S.; Hu, L.; Feuerstein, B.G.; Deen, D.F.

1995-01-01

Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF-126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propodium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose-dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similiar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony-forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors. 22 refs., 5 figs

The effect of ploidy on the modification of the shoulder region of hypoxic cell-survival curves by the biradical, Ro.03-6061

International Nuclear Information System (INIS)

Millar, B.C.; Millar, J.L.

1977-01-01

The biradical nitroxyl, Ro.03-6061, sensitized two lines of mouse L cell to ionizing radiation when the cells were rendered hypoxic. Although the biradical reduced the D 0 value of the hypoxic cell-survival curve in each instance, it had no significant effect on the shoulder region. A hybrid line produced from these two strains was more radioresistant than either parent. In this instance, the biradical suppressed the shoulder region of the hypoxic cell-survival curve, but had no effect on the D 0 value. In a second system, the biradical selectivity sensitized hypoxic cells of a diploid and a tetraploid clone of Syrian hamster cells (BHK21/C15). The survival-curve characteristics of both clones were similar. The biradical reduced the D 0 value but did not significantly change the shoulder region of the hypoxic cell-survival curve. An aneuploid line sub-cultured from the tetraploid clone was much more resistant to radiation. In this instance, there was a decrease in the D 0 value of hypoxic cells in the presence of the biradical, but the extrapolation number was increased to a value similar to that for cells irradiated in air. (author)

The use of drugs which deplete intracellular glutathione in hypoxic cell radiosensitization

International Nuclear Information System (INIS)

Bump, E.A.; Yu, N.Y.; Brown, J.M.

1982-01-01

Diethylmaleate (DEM) is a thiol-biding reagent with specificity toward glutathione. Treatment of chinese hamster ovary (CHO) cells in vitro with 2 x 10 -4 M DEM for one hour results in a decrease in glutathione content to less than 5% of control, without cytotoxicity. This treatment results in dose-modifying sensitization to radiation under hypoxic conditions, with no effect on the shoulder of the radiation survival curve. No effect on the radiation sensitivity of oxygenated cells was seen. DEM pretreatment enhances the radiosensitization of hypoxic cells by misonidazole, as well. Similar results were obtained in vivo with EMT6 tumors in BALB/c mice. Analysis of DNA damage by the alkaline elution assay indicates that DEM enhances radiation-induced single-strand breaks, but does not significantly affect repair, while diamide and N-ethylmaleimide inhibit repair, in addition to enhancing radiation-induced single-strand breaks

Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

2016-08-05

The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba{sup 2+}-sensitive inward rectifier K{sup +} current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 2+}. On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba{sup 2+}. We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca{sup 2+} entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC

In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions)

International Nuclear Information System (INIS)

Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T.; Lagarde, P.; Pooter, C.M.J. de; Chomy, F.

1995-01-01

This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs

Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

Science.gov (United States)

Harrison, Luke R.E.; Micha, Dimitra; Brandenburg, Martin; Simpson, Kathryn L.; Morrow, Christopher J.; Denneny, Olive; Hodgkinson, Cassandra; Yunus, Zaira; Dempsey, Clare; Roberts, Darren; Blackhall, Fiona; Makin, Guy; Dive, Caroline

2011-01-01

Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737–induced cell death. Tumor xenografts in ABT-737–treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1–sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors. PMID:21393866

Superfractionation as a potential hypoxic cell radiosensitizer: prediction of an optimum dose per fraction

International Nuclear Information System (INIS)

Dasu, Alexandru; Denekamp, Juliana

1999-01-01

Purpose: A dose 'window of opportunity' has been identified in an earlier modeling study if the inducible repair variant of the LQ model is adopted instead of the pure LQ model, and if all survival curve parameters are equally modified by the presence or absence of oxygen. In this paper we have extended the calculations to consider survival curve parameters from 15 sets of data obtained for cells tested at low doses using clonogenic assays. Methods and Materials: A simple computer model has been used to simulate the response of each cell line to various doses per fraction in multifraction schedules, with oxic and hypoxic cells receiving the same fractional dose. We have then used pairs of simulated survival curves to estimate the effective hypoxic protection (OER') as a function of the dose per fraction. Results: The resistance of hypoxic cells is reduced by using smaller doses per fraction than 2 Gy in all these fractionated clinical simulations, whether using a simple LQ model, or the more complex LQ/IR model. If there is no inducible repair, the optimum dose is infinitely low. If there is inducible repair, there is an optimum dose per fraction at which hypoxic protection is minimized. This is usually around 0.5 Gy. It depends on the dose needed to induce repair being higher in hypoxia than in oxygen. The OER' may even go below unity, i.e. hypoxic cells may be more sensitive than oxic cells. Conclusions: If oxic and hypoxic cells are repeatedly exposed to doses of the same magnitude, as occurs in clinical radiotherapy, the observed hypoxic protection varies with the fractional dose. The OER' is predicted to diminish at lower doses in all cell lines. The loss of hypoxic resistance with superfractionation is predicted to be proportional to the capacity of the cells to induce repair, i.e. their intrinsic radioresistance at a dose of 2 Gy

Keynote address: cellular reduction of nitroimidazole drugs: potential for selective chemotherapy and diagnosis of hypoxic cells

International Nuclear Information System (INIS)

Chapman, J.D.; Lee, J.; Meeker, B.E.

1989-01-01

Nitroimidazole drugs were initially developed as selective radiosensitizers of hypoxic cells and, consequently, as adjuvants to improve the local control probabilities of current radiotherapies. Misonidazole (MISO), the prototype radiosensitizing drug, was found in Phase I clinical studies to cause dose-limiting neurotoxicities (mainly peripheral neuropathies). MISO was also found to be cytotoxic in the absence of radiation and to covalently bind to cellular molecules, both processes demonstrating rates much higher in hypoxic compared with oxygenated cells. It is likely that neurotoxicity, cellular cytotoxicity and adduct formation results from reactions between reduction intermediates of MISO and cellular target molecules. Spin-offs from radiosensitizer research include the synthesis and characterization of more potent hypoxic cytotoxins and the exploitation of sensitizer-adducts as probes for measuring cellular and tissue oxygen levels. Current developments in hypoxic cell cytotoxin and hypoxic cell marker research are reviewed with specific examples from studies which characterize the cellular reduction of TF-MISO, (1-(2-nitro-1-imidazolyl)-3[2,2,2-trifluoroethoxy]-2-propanol). 45 references

The lifetime of hypoxic human tumor cells

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Durand, Ralph E.; Sham, Edward

1998-01-01

Purpose: For hypoxic and anoxic cells in solid tumors to be a therapeutic problem, they must live long enough to be therapeutically relevant, or else be rapidly recruited into the proliferating compartment during therapy. We have, therefore, estimated lifetime and recruitment rate of hypoxic human tumor cells in multicell spheroids in vitro, or in xenografted tumors in SCID mice. Materials and Methods: Cell turnover was followed by flow cytometry techniques, using antibodies directed at incorporated halogenated pyrimidines. The disappearance of labeled cells was quantified, and verified to be cell loss rather than label dilution. Repopulation was studied in SiHa tumor xenografts during twice-daily 2.5-Gy radiation exposures. Results: The longevity of hypoxic human tumor cells in spheroids or xenografts exceeded that of rodent cell lines, and cell turnover was slower in xenografts than under static growth as spheroids. Human tumor cells remained viable in the hypoxic regions of xenografts for 4-10 days, compared to 3-5 days in spheroids, and 1-3 days for most rodent cells in spheroids. Repopulation was observed within the first few radiation treatments for the SiHa xenografts and, with accumulated doses of more than 10 Gy, virtually all recovered cells had progressed through at least one S-phase. Conclusion: Our results suggest an important difference in the ability of human vs. rodent tumor cells to withstand hypoxia, and raise questions concerning the increased longevity seen in vivo relative to the steady-state spheroid system

Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status

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Sprong, Debbie; Janssen, Hilde L.; Vens, Conchita; Begg, Adrian C.

2006-01-01

Purpose: To determine the role of DNA repair in hypoxic radioresistance. Methods and Materials: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. Results: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. Conclusion: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity

Hypoxic enhancement of exosome release by breast cancer cells

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King, Hamish W; Michael, Michael Z; Gleadle, Jonathan M

2012-01-01

Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. Breast cancer cell lines were cultured under either moderate (1% O 2 ) or severe (0.1% O 2 ) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant. Exposure of three different breast cancer cell lines to moderate (1% O 2 ) and severe (0.1% O 2 ) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions. These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release

Development of a real-time imaging system for hypoxic cell apoptosis

Directory of Open Access Journals (Sweden)

Go Kagiya

2016-01-01

Full Text Available Hypoxic regions within the tumor form due to imbalances between cell proliferation and angiogenesis; specifically, temporary closure or a reduced flow due to abnormal vasculature. They create environments where cancer cells acquire resistance to therapies. Therefore, the development of therapeutic approaches targeting the hypoxic cells is one of the most crucial challenges for cancer regression. Screening potential candidates for effective diagnostic modalities even under a hypoxic environment would be an important first step. In this study, we describe the development of a real-time imaging system to monitor hypoxic cell apoptosis for such screening. The imaging system is composed of a cyclic luciferase (luc gene under the control of an improved hypoxic-responsive promoter. The cyclic luc gene product works as a caspase-3 (cas-3 monitor as it gains luc activity in response to cas-3 activation. The promoter composed of six hypoxic responsible elements and the CMV IE1 core promoter drives the effective expression of the cyclic luc gene in hypoxic conditions, enhancing hypoxic cell apoptosis visualization. We also confirmed real-time imaging of hypoxic cell apoptosis in the spheroid, which shares properties with the tumor. Thus, this constructed system could be a powerful tool for the development of effective anticancer diagnostic modalities.

Induction of cancer cell death by proton beam in tumor hypoxic region

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Hur, T. R.; Lee, Y. M.; Park, J. W.; Sohn, E. J.

2006-05-01

The physical properties of charged particles such as protons are uniquely suited to target the radiation dose precisely in the tumor. In proton therapy, the Bragg peak is spread out by modulating or degrading the energy of the particles to cover a well defined target volume at a given depth. Due to heterogeneity in the various tumors and end-points as well as in the physical properties of the beams considered, it is difficult to fit the various results into a clear general description of the biological effect of proton in tumor therapy. Tumor hypoxia is a main obstacle to radiotherapy, including gamma-ray. Survived tumor cells under hypoxic region are resistant to radiation and more aggressive to be metastasized. To investigate the dose of proton beam to induce cell death of various tumor cells and hypoxic tumor cells at the Bragg peak in vitro, we used 3 kinds of tumor cells, lung cancer, leukemia and hepatoma cells. Proton beam induces apoptosis in Lewis lung carcinoma cells dose dependently and, slightly in leukemia but not in hepatoma cells at all. Above 1000 gray of proton beam, 60% of cells died even the hypoxic cells in Lewis lung carcinoma cells. But the Molt-4 leukemia cells showed milder effect, 20% cell death by the above 1000 Gray of proton beam and typical resistant pattern (5-10%) of hypoxia in desferrioxamine treated cells. Hepatoma cells (HepG2) were not responsive to proton beam even in rather higher dose (4000G). However, by the gamma-irradiation, Molt-4 was more sensitive than hepatoma or lung cancer cells, but still showed hypoxic resistance. The cell death by proton beam in Lewis lung carcinoma cells was confirmed by PARP cleavage and may be mediated by increased p53. Pro-caspases were also activated and cleaved by the proton beam irradiations for lung cancer cell death. In conclusion, high dose of proton beam (above 1000 gray) may be a good therapeutic radiation even in hypoxic region at the Bragg peak, but further investigations about the

Inhibition of glycolysis by misonidazole in hypoxic cells

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Ling, L.; Sutherland, R.

1984-01-01

Inhibition of glycolysis has been postulated to be a mechanism of misonidazole (MISO) toxicity in hypoxic cells. To investigate the effect of MISO on glycolysis, glucose transport and its consumption and lactate formation were measured. Exponential EMT6 cells (10/sup 6/ cells/ml) were made hypoxix by continuous gassing in 3% CO/sub 2/ in N/sub 2/. They were then treated with 5mM MISO for various times, then washed and analysed for their rates of anaerobic glycolysis. Glucose and lactate content were determined enzymatically. The rates of both glucose consumption and lactate formation decreased after 30 min hypoxic incubation with MISO. After 90 min, the rates were not measurable even though the cells still excluded Trypan Blue. There was, however, a parallel decrease in plating efficiency. These data suggest that the inhibition of glycolysis is an important mechanism of hypoxic toxicity of MISO. To locate the site of inhibition, studies were initiated to look at glucose transport by following the uptake of /sup 14/-C-3-0-methyl-glucose, a nonmetabolised glucose analog. Results obtained so far indicate that up to 90 min of hypoxic incubation with MISO, there was no change in the kinetics of the uptake of his analog. Therefore, the results showed that in hypoxic cells treated with MISO, the glucose transport system was unaffected. However, there was a rapid decrease in anaerobic glycolysis

Hypoxic contraction of cultured pulmonary vascular smooth muscle cells

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Murray, T.R.; Chen, L.; Marshall, B.E.; Macarak, E.J.

1990-01-01

The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells

Radiolabeled cypoxic cell sensitizers: tracer for assessment of ischemia

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Mathias, C.J.; Welch, M.J.; Kilbourn, M.R.; Jerabek, P.A.; Patrick, T.B.; Raichle, M.E.; Krohn, K.A.; Rasey, J.S.; Shaw, D.W.

1987-01-01

Hypoxic, non-functional, but viable, tissue may exist in heart and brain following an arterial occlusion. Identification of such tissue in vivo is crucial to the development of effective treatment strategies. It has been suggested that certain compounds capable of sensitizing hypoxic tumor cells to killing by x-rays (i.e., misonidazole) might serve as in vivo markers of hypoxic tissue in ischemic myocardium or brain if properly radiolabeled. To this end the authors have radiolabeled two fluorinated analogs of nitroimidazole based hypoxic cell sensitizers with the 110 minute half-lived positron-emitting fluorine-18. The ability of these tracers to quantitate the presence of hypoxic tissue has been studied in a gerbil stroke model. The in vivo uptake of one of these tracers [F-18]-fluoronormethyoxymisonidazole is dependent on the extent of tissue hypoxia, and thus, appears to have potential as a diagnostic indicator of non-functional but viable tissue when the tracer is used in conjunction with positron emission tomography. 80 references, 2 figures, 1 table

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.

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Christian Frezza

Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

Insulin-like growth factor stimulation increases radiosensitivity of a pancreatic cancer cell line through endoplasmic reticulum stress under hypoxic conditions

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Isohashi, Fumiaki; Endo, Hiroko; Mukai, Mutsuko; Inoue, Masahiro; Inoue, Takehiro

2008-01-01

Tumor hypoxia is an obstacle to radiotherapy. Radiosensitivity under hypoxic conditions is determined by molecular oxygen levels, as well as by various biological cellular responses. The insulin-like growth factor (IGF) signaling pathway is a widely recognized survival signal that confers radioresistance. However, under hypoxic conditions the role of IGF signaling in radiosensitivity is still poorly understood. Here, we demonstrate that IGF-II stimulation decreases clonogenic survival under hypoxic conditions in the pancreatic cancer cell lines AsPC-1 and Panc-1, and in the human breast cancer cell line MCF-7. IGF treatment under hypoxic conditions suppressed increased radiation sensitivity in these cell lines by pharmacologically inhibiting the phosphoinositide 3-kinase-mammalian target of rapamycin pathway, a major IGF signal-transduction pathway. Meanwhile, IGF-II induced the endoplasmic reticulum stress response under hypoxia, including increased protein levels of CHOP and ATF4, mRNA levels of CHOP, GADD34, and BiP as well as splicing levels of XBP-1. The response was suppressed by inhibiting phosphoinositide 3-kinase and mammalian target of rapamycin activity. Overexpression of CHOP in AsPC-1 cells increased radiation sensitivity by IGF-II simulation under hypoxic conditions, whereas suppression of CHOP expression levels with small hairpin RNA or a dominant negative form of a proline-rich extensin-like receptor protein kinase in hypoxia decreased IGF-induced radiosensitivity. IGF-induced endoplasmic reticulum stress contributed to radiosensitization independent of cell cycle status. Taken together, IGF stimulation increased radiosensitivity through the endoplasmic reticulum stress response under hypoxic conditions. (author)

Comparison of radiation-induced DNA-protein cross-links formed in oxic, hypoxic, and glutathione depleted cells

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Xue, L.; Friedman, L.R.; Chiu, S.; Ramakrishnan, N.; Oleinick, N.L.

1987-01-01

Treatment of cells with L-buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH). Subsequent metabolism depletes the cells of GSH. GSH-depletion sensitizes both oxic and hypoxic cells to the lethal effects of ionizing radiation. DNA-protein cross-links (DPC) are formed preferentially between DNA sequences active in transcription and a subset of proteins of the nuclear matrix. Thus, DPC may be an indicator lesion of damage in sensitive regions of the genome. The interrelationships between GSH level, oxic vs. hypoxic status, and the yield of DPC have been studied in terms of number of lesions and repair rate in Chinese hamster V79 and in human lung carcinoma A549 cells. The data suggest that elevated background levels of DPC are indicative of a reduced repair capacity, and greater radiation-induced yields of DPC in hypoxia may also be indicative of a compromised repair mechanism

Cell-cycle distributions and radiation responses of Chinese hamster cells cultured continuously under hypoxic conditions

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Tokita, N.; Carpenter, S.G.; Raju, M.R.

1984-01-01

Cell-cycle distributions were measured by flow cytometry for Chinese hamster (CHO) cells cultured continuously under hypoxic conditions. DNA histograms showed an accumulation of cells in the early S phase followed by a traverse delay through the S phase, and a G 2 block. During hypoxic culturing, cell viability decreased rapidly to less than 0.1% at 120 h. Radiation responses for cells cultured under these conditions showed an extreme radioresistance at 72 h. Results suggest that hypoxia induces a condition similar to cell synchrony which itself changes the radioresistance of hypoxic cells. (author)

Irradiation combined with Bleomycin treatment of synchronized cells in culture under oxic and hypoxic conditions

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Midander, J.; Littbrand, B.; Edsmyr, F.

1980-01-01

Bleomycin-treated cells are sensitized to radiation delivered under oxic conditions both in the early S and G 2 phases of the cycle, irradiated under hypoxic conditions, sensitization occurs only in the early S phase. This difference in the sensitizing effect of the drug is discussed in regard to the possible clinical advantages of a combined treatment of tumours with irradiation and Bleomycin. (Auth.)

Enhanced induction of SCEs in hypoxic mammalian cells by ionizing radiation

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Tofilon, P.J.; Meyn, R.E.

1985-01-01

Ionizing radiation is, in general, a poor inducer of sister chromatoid exchanges (SCEs). However, the authors previously observed an increase in X-ray induced DNA-protein crosslinks in hypoxic cells, as compared to aerated cells, suggesting that in the absence of oxygen, X rays induce a qualitatively different DNA lesion. Therefore, they examined the effect of X-rays on SCE induction under hypoxic conditions. CHO cells were rendered hypoxic by incubation at 37 0 for 3 hr. in evacuated glass ampules and irradiated with graded doses of X-rays. After irradiation, cells were incubated in medium containing BrdUrd and the SCE assay performed. At each dose tested (0-900 rads) the number of SCEs induced by X-rays in hypoxic cells was approximately 2.5 fold the number induced in aerated cells. When a 16-hr. repair-incubation interval was allowed between irradiation and BrdUrd labeling, the number of SCEs returned to background levels. In further experiments, repair-deficient cells, incapable of completely removing crosslinks from their DNA, did not completely restore SCE levels to background within the repair period. These data provide further evidence suggesting that hypoxic cells respond differently to radiation in a qualitative sense, in addition to the well known quantitative sense

Fabrication and characterization of UV-emitting nanoparticles as novel radiation sensitizers targeting hypoxic tumor cells

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Squillante, Michael R.; Jüstel, Thomas; Anderson, R. Rox; Brecher, Charles; Chartier, Daniel; Christian, James F.; Cicchetti, Nicholas; Espinoza, Sara; McAdams, Daniel R.; Müller, Matthias; Tornifoglio, Brooke; Wang, Yimin; Purschke, Martin

2018-06-01

Radiation therapy is one of the primary therapeutic techniques for treating cancer, administered to nearly two-thirds of all cancer patients. Although largely effective in killing cancer cells, radiation therapy, like other forms of cancer treatment, has difficulty dealing with hypoxic regions within solid tumors. The incomplete killing of cancer cells can lead to recurrence and relapse. The research presented here is investigating the enhancement of the efficacy of radiation therapy by using scintillating nanoparticles that emit UV photons. UV photons, with wavelengths between 230 nm and 280 nm, are able to inactivate cells due to their direct interaction with DNA, causing a variety of forms of damage. UV-emitting nanoparticles will enhance the treatment in two ways: first by generating UV photons in the immediate vicinity of cancer cells, leading to direct and oxygen-independent DNA damage, and second by down-converting the applied higher energy X-rays into softer X-rays and particles that are more efficiently absorbed in the targeted tumor region. The end result will be nanoparticles with a higher efficacy in the treatment of hypoxic cells in the tumor, filling an important, unmet clinical need. Our preliminary experiments show an increase in cell death using scintillating LuPO4:Pr nanoparticles over that achieved by the primary radiation alone. This work describes the fabrication of the nanoparticles, their physical characterization, and the spectroscopic characterization of the UV emission. The work also presents in vitro results that demonstrate an enhanced efficacy of cell killing with x-rays and a low unspecific toxicity of the nanoparticles.

Ultrastructural alterations in hypoxic EMT-6/RO cells treated with misonidazole

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Wilbur, D.C.; Mulcahy, R.T.

1984-01-01

Ultrastructural alterations in hypoxic EMT-6 tumor cells were quantitatively analyzed as a function of time in the presence and absence of 1.0mM MISO. Control and MISO-treated monolayer cultures were maintained in hypoxic chambers at 37 0 C. At intervals after initiation of hypoxia, the cells were fixed and prepared for electron microscopy. The major ultrastructural alterations observed in untreated and MISO-treated hypoxic cells included mitochondrial swelling and accumulation of cytoplasmic lipid vacuoles. Mean mitochondrial area and relative cytoplasmic area occupied by lipid vacuoles were determined morphometrically. Mitochondrial damage was also scored qualitatively based on distortions in configuration. In the absence of MISO both parameters of mitochondrial injury increased over a period of two hours, after which little further change was noted. A progressive increase in lipid vacuolization was also seen. In the presence of MISO, mitochondrial swelling and lipid vacuole formation were significantly increased. The proportion of irreversibly damaged mitochondria was markedly enhanced. MISO treatment also accelerated the expression of these changes. The accelerated expression of hypoxic-related injury in MISO treated cells suggests that cytotoxicity is related to accentuation of hypoxic injury, perhaps by inhibition of glycolysis

Relative survival of hybrid x-ray-resistant, and normally sensitive mammalian cells exposed to x rays and protons under aerobic and hypoxic conditions

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Williams, J.R.; Gould, R.G.; Flynn, D.; Robertson, J.B.; Little, J.B.

1978-01-01

Survival of an x-ray-resistant hybrid cell line (HD 1 ) and a normally responsive cell line (H 4 ) have been compared when irradiated under induced hypoxia by both protons and X rays. The two cell lines are similarly protected when irradiated under hypoxic conditions with oxygen enhancement ratios of 2.8 and 2.7, respectively. The protection is consistent with a dose-modifying factor. No statistically significant difference is observed between cell inactivation by x rays and protons in either cell line, whether irradiated under aerobic or hypoxic conditions

AT-406, an IAP inhibitor, activates apoptosis and induces radiosensitization of normoxic and hypoxic cervical cancer cells.

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Lu, Jing; Qin, Qin; Zhan, Liang-Liang; Liu, Jia; Zhu, Hong-Cheng; Yang, Xi; Zhang, Chi; Xu, Li-Ping; Liu, Zhe-Ming; Wang, Di; Cui, He-Qing; Meng, Ciu-Ciu; Cai, Jing; Cheng, Hong-Yan; Sun, Xin-Chen

2014-01-01

IAP antagonists increased the antitumor efficacy of X-irradiation in some types of cancers, but their effects on hypoxic cancer cells remain unclarified. We aims to investigate the radiosensitizing effect of an IAP inhibitor AT-406 on cervical cancer cell lines under both normoxia and hypoxia conditions. Hela and Siha cells were treated to investigate the effects of drug administration on cell proliferation, apoptosis, and radiosensitivity. Western blot analysis was used to determine the role of AT-406 in inhibition of IAPs. The pathway of apoptosis was characterized by caspases activity assay. AT-406 potently sensitized Hela cells but not Siha cells to radiation under normoxia. Notably, the radiosensitizing effect of AT-406 on hypoxic cells was more evident than on normoxic cells in both cell lines. Further mechanism studies by western blot showed that under normoxia AT-406 decreased the level of cIAP1 in Hela cells in a dose-dependent manner; while additional downregulation of XIAP expression was induced by AT-406 treatment under hypoxia in both cell lines. Finally, AT-406 works on both extrinsic death receptor and intrinsic mitochondrial apoptosis pathways to activate apoptosis. Totally, AT-406 acts as a strong radiosensitizer in human cervical cancer cells, especially in hypoxic condition.

New strategy of cancer therapy by targeting the hypoxic circumstances

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Yasui, Hironobu; Yamamori, Tohru; Meike, Shunsuke; Eitaki, Masato; Kuwabara, Mikinori; Inanami, Osamu; Iizuka, Daisuke

2010-01-01

Described are studies on the sensitization of tumor cells in hypoxic circumstances (known as radio-resistant cells) by authors' recent molecular targeting to adaptive response as well as by the usual agents like nitro-imidazole compounds, and on the intermittent hypoxia, a new topic in this field. The hypoxia-inducible factor-1 (HIF-1) is a transcriptional factor and has been known to activate its many downstream genes to cause adoptive response of hypoxic cells. Authors have studied the anti-tumor and radiation sensitizing effects of ethynyl-cytidine (EC) which is found to suppress RNA synthesis through cytidine kinase (CK) inhibition, and the compound is of specificity to tumor cells as they have 5-10 times higher CK activity than normal cells. Authors have also found that EC is of the sensitizing efficacy to normoxic and hypoxic cells by enhancing the radiation-induced apoptosis essentially through inhibition of HIF-1 expression. Intermittent hypoxia in the tumor which has characteristic abnormal vascular morphology and function, occurs by the transient reduction of blood flow and occlusion of vessels in the tissue within minute to hour time cycles. Little is known about the regional hypoxic region and its distribution in the tumor due to difficulty of their detection and quantification. For this, authors have measured the temporal changes of oxygen levels in the mouse tumor with triaryl methyl radical, an oxygen-sensitive contrast compound continuously injected, by microwave-pulsed electron spin resonance imaging (EPRI). By superimposing the EPRI and T2-weighted MRI, the oxymetric imaging is possible in the tumor, which reveals the difference of oxygen level variation depending on the cell type and tissue size. Findings in the field are expected to give important information for more effective cancer therapy and its prognostic prediction in future. (T.T.)

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

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

2017-01-01

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

Apoptosis, energy metabolism, and fraction of radiobiologically hypoxic cells: a study of human melanoma multicellular spheroids.

Science.gov (United States)

Rofstad, E K; Eide, K; Skøyum, R; Hystad, M E; Lyng, H

1996-09-01

The magnitude of the fraction of radiobiologically hypoxic cells in tumours is generally believed to reflect the efficiency of the vascular network. Theoretical studies have suggested that the hypoxic fraction might also be influenced by biological properties of the tumour cells. Quantitative experimental results of cell energy metabolism, hypoxia- induced apoptosis, and radiobiological hypoxia are reported here. Human melanoma multicellular spheroids (BEX-c and WIX-c) were used as tumour models to avoid confounding effects of the vascular network. Radiobiological studies showed that the fractions of hypoxic cells in 1000-microM spheroids were 32 +/- 12% (BEX-c) and 2.5 +/- 1.1% (WIX-c). The spheroid hypoxic volume fractions (28 +/- 6% (BEX-c) and 1.4 +/- 7% (WIX-c)), calculated from the rate of oxygen consumption per cell, the cell packing density, and the thickness of the viable rim, were similar to the fractions of radiobiologically hypoxic cells. Large differences between tumours in fraction of hypoxic cells are therefore not necessarily a result of differences in the efficiency of the vascular network. Studies of monolayer cell cultures, performed to identify the biological properties of the BEX-c and WIX-c cells leading to this large difference in fraction of hypoxic cells, gave the following results: (1) WIX-c showed lower cell surviving fractions after exposure to hypoxia than BEX-c, (2) WIX-c showed higher glucose uptake and lactate release rates than BEX-c both under aerobic and hypoxic conditions, and (3) hypoxia induced apoptosis in WIX-c but not in BEX-c. These observations suggested that the difference between BEX-c and WIX-c spheroids in fraction of hypoxic cells resulted partly from differences in cell energy metabolism and partly from a difference in capacity to retain viability under hypoxic stress. The induction of apoptosis by hypoxia was identified as a phenomenon which has an important influence on the magnitude of the fraction of

Effects of oxygen on intrinsic radiation sensitivity: A test of the relationship between aerobic and hypoxic linear-quadratic (LQ) model parameters

International Nuclear Information System (INIS)

Carlson, David J.; Stewart, Robert D.; Semenenko, Vladimir A.

2006-01-01

The poor treatment prognosis for tumors with high levels of hypoxia is usually attributed to the decreased sensitivity of hypoxic cells to ionizing radiation. Mechanistic considerations suggest that linear quadratic (LQ) survival model radiosensitivity parameters for hypoxic (H) and aerobic (A) cells are related by α H =α A /oxygen enhancement ratio (OER) and (α/β) H =OER(α/β) A . The OER parameter may be interpreted as the ratio of the dose to the hypoxic cells to the dose to the aerobic cells required to produce the same number of DSBs per cell. The validity of these expressions is tested against survival data for mammalian cells irradiated in vitro with low- and high-LET radiation. Estimates of hypoxic and aerobic radiosensitivity parameters are derived from independent and simultaneous least-squares fits to the survival data. An external bootstrap procedure is used to test whether independent fits to the survival data give significantly better predictions than simultaneous fits to the aerobic and hypoxic data. For low-LET radiation, estimates of the OER derived from the in vitro data are between 2.3 and 3.3 for extreme levels of hypoxia. The estimated range for the OER is similar to the oxygen enhancement ratios reported in the literature for the initial yield of DSBs. The half-time for sublethal damage repair was found to be independent of oxygen concentration. Analysis of patient survival data for cervix cancer suggests an average OER less than or equal to 1.5, which corresponds to a pO 2 of 5 mm Hg (0.66%) in the in vitro experiments. Because the OER derived from the cervix cancer data is averaged over cells at all oxygen levels, cells irradiated in vivo under extreme levels of hypoxia (<0.5 mm Hg) may have an OER substantially higher than 1.5. The reported analyses of in vitro data, as well as mechanistic considerations, provide strong support for the expressions relating hypoxic and aerobic radiosensitivity parameters. The formulas are also useful

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

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Shima, Haruko; Takubo, Keiyo; Iwasaki, Hiroko; Yoshihara, Hiroki; Gomei, Yumiko; Hosokawa, Kentaro; Arai, Fumio; Takahashi, Takao; Suda, Toshio

2009-01-01

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ null (NOG) mice. Hypoxic culture (1% O 2 ) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34 + CD38 - cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.

Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

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Bache, Matthias; Taubert, Helge; Vordermark, Dirk; Zschornak, Martin P; Passin, Sarina; Keßler, Jacqueline; Wichmann, Henri; Kappler, Matthias; Paschke, Reinhard; Kaluđerović, Goran N; Kommera, Harish

2011-01-01

Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses. Under normoxic conditions, a half maximal inhibitory concentration (IC 50 ) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions. Our results suggest that BA is capable

Intervention of oxygen-control ability to radiation sensitivity, cell aging and cell transformation

International Nuclear Information System (INIS)

Yoshii, Hanako; Watanabe, Masami

2009-01-01

Oxygen is essential for life, and cells have therefore developed numerous adaptive responses to oxygen change. Here, we examined the difference in oxygen-control functions of human (HE), mouse (ME), and Syrian hamster embryo (SHE) cells cultured under different oxygen conditions (0.5%, 2% and 20%), and also examined whether oxygen tensions contributed to cellular lifespan and transformation. HE cells had their replicative lifespan slightly extended under hypoxic (0.5% and 2% oxygen) conditions, but were not immortalized under any of the oxygen concentrations. On the other hand, although ME cells cultured under 20% oxygen tension decreased their proliferation potency temporarily at early stage, all rodent cells were immortalized and acquired anchorage-independency, regardless of oxygen tension. These results suggest that cellular oxygen control function is related to sensitivities cellular immortalization and transformation. To understand intervention of oxygen control ability on cellular immortalization and transformation, we examined the intracellular oxidative level, mitochondria functions and radiation sensitivity. Intracellular oxidative levels of hypoxically cultured rodent cells were significantly enhanced. Mitochondrial membrane potential was altered depend on oxygen tensions, but the change was not parallel to mitochondria number in rodent cells. ME cells were particularly sensitive to oxygen change, and showed a clear oxygen effect on the X-ray survival. However, there was no difference in frequency of radiation-induced micronuclei between HE and ME cells. These results suggest that the response to oxygen change differs markedly in HE and rodent cells. (author)

CHANGES IN THE GLUTATHIONE SYSTEM IN P19 EMBRYONAL CARCINOMA CELLS UNDER HYPOXIC CONDITIONS

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D. S. Orlov

2015-01-01

Full Text Available Introduction. According to modern perceptions, tumor growth, along with oxidative stress formation, is accompanied by hypoxia. Nowadays studying the regulation of cellular molecular system functioning by conformational changes in proteins appears to be a topical issue. Research goal was to evaluate the state of the glutathione system and the level of protein glutathionylation in P19 embryonal carcinoma (EC cells under hypoxic conditions.Material and methods. P19 EC cells (mouse embryonal carcinoma cultured under normoxic and hypox-ic conditions served the research material.The concentration of total, oxidized, reduced and protein-bound glutathione, the reduced to oxidized thiol ratio as well as glutathione peroxidase and glutathione reductase activity were determined by spectropho-tometry.Results. Glutathione imbalance was accompanied by a decrease in P19 EC cell redox status under hypox-ic conditions against the backdrop of a rise in protein-bound glutathione.Conclusions. As a result of the conducted study oxidative stress formation was identified when modeling hypoxia in P19 embryonal carcinoma cells. The rise in the concentration of protein-bound glutathione may indicate the role of protein glutathionylation in regulation of P19 cell metabolism and functions un-der hypoxia. 

Further evidence for the absence of a hypoxic fraction in the 9L rat tumour multicellular spheroid system

International Nuclear Information System (INIS)

Gutin, P.H.; Barcellos, M.H.; Shrieve, D.C.; Sano, Y.; Bernstein, M.; Deen, D.F.

1982-01-01

The 9L gliosarcoma is an N-methylnitrosourea-induced rat brain tumour that has served as a predictive model for the efficacy of various chemotherapeutic agents against human brain tumours. Because it is one of two known animal tumour models that has no hypoxic fraction, the 9L model is of questionable value for the study of the radiobiology of hypoxic cell sensitizers. Hypoxic 9L monolayer cells are sensitive to misonidazole, as shown by the abrupt decrease in survival after a 2-4 h radiation exposure. However, when 9L spheroids in the size ranges of 200-300, 300-400, 500-600 and 1027+-33μm were incubated in euoxic spinner culture for up to 96 h in 1.5 or 3.0 mM misonidazole, there was no effect on the survival of the dissociated cells over a dose range 0-20 Gy. It is concluded that, in view of the demonstrated sensitivity to misonidazole of hypoxic 9L cells in monolayer culture, this finding provides further evidence that there are no hypoxic cells even in large 9L spheroids with a histologically distinct zone of central necrosis. Moreover, 9L spheroids irradiated in the presence of 3.0 mM misonidazole showed no dose enhancement. (U.K.)

Interstitial administration of perfluorochemical emulsions for reoxygenation of hypoxic tumor cells

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Woo, D.V.; Seegenschmiedt, H.; Schweighardt, F.K.; Emrich, J.; McGarvey, K.; Caridi, M.; Brady, L.W.

1987-01-01

Microparticulate perfluorochemical (PFC) emulsions have the capacity to solubilize significant quantities of oxygen compared to water. Although systemic administration of such emulsions may enhance oxygen delivery to some tissues, hypoxic tumor cells have marginal vascular supplies. The authors report studies which directly attempt to oxygenate hypoxic tumor cells by interstitial administration of oxygenated PFC emulsions followed by radiation therapy. Fortner MMI malignant melanomas (21 day old) grown in Syrian Golden hamsters were injected directly with either oxygenated PFC emulsions or Ringers solution. The volume of test substance administered was equal to 50% of the tumor volume. The tumors were immediately irradiated with 25 Gy of 10 MeV photons (Clinac 18). The tumor dimensions were measured daily post irradiation and the tumor doubling time determined. The results suggest that interstitial administration of oxygenated PFC emulsions directly into tumors followed by radiation therapy may increase the likelihood of killing hypoxic tumor cells

Inhibition of Siah2 Ubiquitin Ligase by Vitamin K3 Attenuates Chronic Myeloid Leukemia Chemo-Resistance in Hypoxic Microenvironment.

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Huang, Jixian; Lu, Ziyuan; Xiao, Yajuan; He, Bolin; Pan, Chengyun; Zhou, Xuan; Xu, Na; Liu, Xiaoli

2018-02-05

BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.

The effect of radiosensitizers on the survival response of hypoxic mammalian cells: The low X-ray dose region, hypersensitivity and induced radioresistance

International Nuclear Information System (INIS)

Skov, K.A.; MacPhail, H.S.; Marples, B.

1994-01-01

It has been shown previously that the extent of chemical modification of the hypoxic radiation response is dependent on dose. Some types of sensitizer are more effective at low doses (to 4 Gy) than at higher doses. Since such drugs are possible adjuvants to radiotherapy, the mechanisms responsible for the variable response at clinical doses are summarized, and the effects of cisplatin and buthionine sulfoximine on the purported induced response to radiation in hypoxic cells are presented. Cisplatin at a low, nontoxic concentration (1 μM) appears to abolish the increased radioresistant portion of the survival response. A role for high-mobility-group protein binding by platinum drugs is hypothesized to explain their interaction with radiation, and conversely, it is suggested that the heretofore unexplained different behavior of certain hypoxic sensitizers at low doses could be, at least in part, an effect on the induction of resistance. 36 refs., 2 figs

Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis.

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Nanduri, Jayasri; Makarenko, Vladislav; Reddy, Vaddi Damodara; Yuan, Guoxiang; Pawar, Anita; Wang, Ning; Khan, Shakil A; Zhang, Xin; Kinsman, Brian; Peng, Ying-Jie; Kumar, Ganesh K; Fox, Aaron P; Godley, Lucy A; Semenza, Gregg L; Prabhakar, Nanduri R

2012-02-14

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

Reoxygenation of hypoxic cells by tumor shrinkage during irradiation. A computer simulation

International Nuclear Information System (INIS)

Kocher, M.; Treuer, H.

1995-01-01

A 3-dimensional computer simulation was developed in order to estimate the impact of tumor shrinkage on reoxygenation of chronic hypoxic tumor cells during a full course of fractionated irradiation. The growth of a small tumor situated in a vascularized stroma with 350 capillary cross-sections/mm 3 which were displaced by the growing tumor was simulated. Tumors contained 10 4 cells when irradiation started, intrinsic radiosensitivity was set to either low (α=0.3 Gy -1 , β=0.03 Gy -2 ) or high (α=0.4 Gy -1 , β=0.04 Gy -2 ) values. Oxygen enhancement ratio was 3.0, potential tumor doubling time T pot =1, 2 or 5 days. A simulated fractionated radiotherapy was carried out with daily fractions of 2.0 Gy, total dose 50 to 70 Gy. The presence or absence of factors preventing tumor cord shrinkage was also included. During the growth phase, all tumors developed a necrotic core with a hypoxic cell fraction of 25% under these conditions. During irradiation, the slower growing tumors (T pot =2 to 5 days) showed complete reoxygenation of the hypoxic cells after 30 to 40 Gy independent from radiosensitivity, undisturbed tumor shrinkage provided. If shrinkage was prevented, the hypoxic fraction rose to 100% after 30 to 50 Gy. Local tumor control, defined as the destruction of all clonogenic and hypoxic tumor cells increased by 20 to 100% due to reoxygenation and 50 Gy were enough in order to sterilize the tumors in these cases. In the fast growing tumors (T pot =1 day), reoxygenation was only observed in the case of high radiosensitivity and undisturbed tumor shrinkage. In these tumors reoxygenation increased the control rates by up to 60%. (orig./MG) [de

The fate of hypoxic (pimonidazole-labelled) cells in human cervix tumours undergoing chemo-radiotherapy

International Nuclear Information System (INIS)

Durand, Ralph E.; Aquino-Parsons, Christina

2006-01-01

Background and purpose: A subset of patients in a clinical study where sequential biopsies were to be obtained during multifraction radiotherapy received pimonidazole prior to initiating treatment, allowing a unique opportunity of following hypoxic cells in situ during therapy. Material and methods: After institutional ethics review and with informed consent, women expecting to undergo radical treatment for cancer of the cervix received pimonidazole hydrochloride, with a biopsy approximately 24 h later. Therapy was then started, and weekly biopsies were obtained. In the laboratory, the biopsies were reduced to single cell suspensions for flow cytometry analysis of DNA content, pimonidazole, and proliferation markers. Results: Pre-treatment pimonidazole-positive cells were largely in G /G 1 . Pimonidazole-labelled cells, though expected to be radioresistant, were markedly decreased even early into treatment, and continued to disappear with a half-time of about 3 days. Concurrently, the cell cycle distribution of the previously hypoxic cells changed from predominantly quiescent to mostly proliferating. Conclusions: While a part of the rapid apparent loss of hypoxic cells was certainly due to loss of pimonidazole adducts through repair and dilution by cell division, the speed with which this occurred suggests that many labelled cells could rapidly re-enter the proliferative pool, a result consistent with many of those pimonidazole-labelled human cervix tumour cells being cyclically, rather than continuously, hypoxic

Imaging and Targeting of Hypoxic Tumor Cells with Use of HIF-1-2

International Nuclear Information System (INIS)

Kizaka-Kondoh, Shinae; Harada, Hiroshi; Tanaka, Shotaro; Hiraoka, Masahiro

2006-01-01

This paper describes imaging (visualization) of transplanted tumor cells under hypoxia in vivo and molecular targeting to kill those cells by inducing their apoptosis. HIF (hypoxia inducible factor) concerned with angiogenesis is induced specifically in hypoxic tumor cells and its activity can be visualized by transfection of reporter vector construct of fluorescent protein GFP or luciferase. Authors established the transfected tumor cells with the plasmid p5HRE-luciferase and when transplanted in the nude mouse, those cells emitted light dependently to their hypoxic conditions, which could be visualized by in vivo imaging system (IVIS) with CCD camera. Authors prepared the oxygen-dependent degradation-procaspase 3-fusion protein (TOP3) to target the hypoxic tumor cells for enhancing their apoptotic signaling, whose apoptosis was actually observed by the IVIS. Reportedly, radiation transiently activates HIF-1 and combination treatment of radiation and TOP3 resulted in the enhanced death of tumor cells. Interestingly, the suppression of tumor growth lasted longer than expected, probably due to inhibition of angiogenesis. Authors called this anti-tumor strategy as the micro-environmental targeting. (T.I.)

Characteristics of fluorinated nitroazoles as hypoxic cell radiosensitizers

International Nuclear Information System (INIS)

Shibamoto, Y.; Nishimoto, S.; Shimokawa, K.

1989-01-01

Types of 2-nitroimidazoles and 3-nitro-1,2,4-triazoles bearing one or two fluorine atoms on their side chains were synthesized to evaluate their physicochemical properties, radiosensitizing effects, and toxicity. The reduction potential of the compounds containing one fluorine was similar to that of misonidazole (MISO), whereas that of the difluorinated compounds was slightly higher. Both mono- and difluorinated compounds had an in vitro sensitizing activity comparable to or slightly higher than that of MISO. The fluorinated 3-nitrotriazoles were almost as efficient as the 2-nitroimidazoles with the same substituent. In vivo, some of the compounds were up to twice more efficient than MISO, whereas others were as efficient as MISO. Toxicity in terms of LD50/7 in mice was quite variable depending on the side-chain structure; the amide derivatives were less toxic than MISO, whereas the alcohol and ether derivatives were more toxic. In view of the radiosensitizing effect and toxicity in vivo, at least one compound, KU-2285 (a 2-nitroimidazole with an N1-substituent of: CH2CF2CONHCH2CH2OH) has been found to be as useful a hypoxic cell sensitizer as SR-2508

Overendocytosis of gold nanoparticles increases autophagy and apoptosis in hypoxic human renal proximal tubular cells

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Ding F

2014-09-01

Full Text Available Fengan Ding,1 Yiping Li,1 Jing Liu,1 Lei Liu,1 Wenmin Yu,1 Zhi Wang,1 Haifeng Ni,2 Bicheng Liu,2 Pingsheng Chen1,2 1School of Medicine, Southeast University, Nanjing, People’s Republic of China; 2Institute of Nephrology, The Affiliated Zhongda Hospital, Southeast University, Nanjing, People’s Republic of China Background: Gold nanoparticles (GNPs can potentially be used in biomedical fields ranging from therapeutics to diagnostics, and their use will result in increased human exposure. Many studies have demonstrated that GNPs can be deposited in the kidneys, particularly in renal tubular epithelial cells. Chronic hypoxic is inevitable in chronic kidney diseases, and it results in renal tubular epithelial cells that are susceptible to different types of injuries. However, the understanding of the interactions between GNPs and hypoxic renal tubular epithelial cells is still rudimentary. In the present study, we characterized the cytotoxic effects of GNPs in hypoxic renal tubular epithelial cells.Results: Both 5 nm and 13 nm GNPs were synthesized and characterized using various biophysical methods, including transmission electron microscopy, dynamic light scattering, and ultraviolet–visible spectrophotometry. We detected the cytotoxicity of 5 and 13 nm GNPs (0, 1, 25, and 50 nM to human renal proximal tubular cells (HK-2 by Cell Counting Kit-8 assay and lactate dehydrogenase release assay, but we just found the toxic effect in the 5 nm GNP-treated cells at 50 nM dose under hypoxic condition. Furthermore, the transmission electron microscopy images revealed that GNPs were either localized in vesicles or free in the lysosomes in 5 nm GNPs-treated HK-2 cells, and the cellular uptake of the GNPs in the hypoxic cells was significantly higher than that in normoxic cells. In normoxic HK-2 cells, 5 nm GNPs (50 nM treatment could cause autophagy and cell survival. However, in hypoxic conditions, the GNP exposure at the same condition led to the

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells

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Johansson, Elinn; Grassi, Elisa S.; Pantazopoulou, Vasiliki

2017-01-01

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell...... marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature...... correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic...

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.

Radiosensitization of hypoxic bacterial cells by nitroimidazoles of low lipophilicity: steady-state and rapid-mix studies

International Nuclear Information System (INIS)

Anderson, R.F.; Patel, K.B.; Sehmi, D.S.

1981-01-01

Radiosensitization of hypoxic bacterial cells by five 2-nitroimidazoles, with similar reduction potentials to misonidazole but having lower lipophilicites, has been measured in Escherichia coli AB 1157 and Streptococcus lactis 712. Sensitization efficiency progressively decreased with decreasing lepophilicity in E. coli but not in S. lactis. This difference is discussed in terms of the differing membrane properties of the two bacteria; E. coli resembled a multicompartment model, as would also be expected with mammalian cells. Rapid-mix experiments are described which show that the radiosensitization observed after experiments are described which show that the radiosensitization observed after preirradiation contact times between ca. 3 and 30 msec is dependent on the lipophilicity of the sensitizer, higher lipophilicity resulting in a lower contact time being required for radiosensitization. This result and the observation that a highly lipophilic compound affects only half the full oxygen enhancement level after short contact times suggest that part of the sensitization process occurs in a lipophilic compartment of the cell

Selective toxicity of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide toward hypoxic mammalian cells

International Nuclear Information System (INIS)

Rauth, A.M.; Mohindra, J.K.

1981-01-01

The chemotherapeutic agent 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) is used in the treatment of malignant melanoma where response rates of 15 to 30% have been reported. Some current interest exists in combining DTIC chemotherapy with localized high-dose (800 rads)-per-fraction radiotherapy in the treatment of unresectable metastatic melanoma. The present work investigates the radiosensitizing and chemotherapeutic properties of DTIC in an in vitro system using Chinese hamster ovary or HeLa cells and in vivo, using the KHT transplantable murine tumor. No evidence of a radiosensitizing effect of DTIC was found towards hypoxic or aerobic cells either in vitro or in vivo. In vitro, high drug concentrations (1 mg/ml) were approximately 5 times more effective in killing hypoxic Chinese hamster ovary or HeLa cells than in killing aerobic cells over exposure times of 0 to 12 hr. The degree of toxicity was drug dose and temperature dependent but was not highly dependent on cell number or cell type. In vivo plasma levels of DTIC were measured with high-pressure liquid chromatography after i.p. injection of drug into C3H mice. At the highest drug doses tested, near the 50% lethal dose in mice for DTIC (0.5 mg/g), the drug was toxic to both aerobic and hypoxic tumor cells with some evidence of increased toxicity towards hypoxic cells. The present work suggests that DTIC may be more efficiently activated under hypoxic conditions as compared to aerobic conditions. The increased toxicity of DTIC under hypoxic versus aerobic conditions may prove to be a feature of this drug that can be exploited in its clinical use and in the design of new analogs of DTIC

Lethal Effect of Thermal Neutrons on Hypoxic Elirlich Ascites Tumour Cells in vitro

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MITSUHIKO, AKABOSHI; KENICHI, KAWAI; HIROTOSHI, MAKI; Research Reactor Institute, Kyoto University; Research Reactor Institute, Kyoto University; Research Reactor Institute, Kyoto University

1985-01-01

Ehrlich ascites tumour cells were irradiated in vitro with thermal neutrons under aerobic and hypoxic conditions, and the survival of their reproductive capacity was assayed in vivo. Only a slight hypoxic protection was observed for thermal neutron irradiation with an oxygen enhancement ratio (OER) of 1.2, as compared with OER of 3.3 for ^Co-γ-rays. Absorbed dose of thermal neutrons was calculated by assuming that the energies of recoiled nuclei were completely absorbed within a cell nucleus....

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction

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Yihua Liu

2017-11-01

Full Text Available Background: The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs. This study sought to address (i the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI in rats. Methods: rat BMSCs were harvested and cultured in normoxic (21% O2, n=27 or hypoxic conditions (5% O2, n=27 until Passage 4 (P4. Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6 or hypoxia (n=6 were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG positron emission tomography (PET imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Results: Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Conclusion: Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction.

Science.gov (United States)

Liu, Yihua; Yang, Xiaoxi; Maureira, Pablo; Falanga, Aude; Marie, Vanessa; Gauchotte, Guillaume; Poussier, Sylvain; Groubatch, Frederique; Marie, Pierre-Yves; Tran, Nguyen

2017-01-01

The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs). This study sought to address (i) the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii) the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI) in rats. rat BMSCs were harvested and cultured in normoxic (21% O2, n=27) or hypoxic conditions (5% O2, n=27) until Passage 4 (P4). Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6) or hypoxia (n=6) were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG) positron emission tomography (PET) imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting angiogenesis. © 2017 The Author(s). Published by S. Karger AG

The hypoxic tumour cell in radiation therapy

International Nuclear Information System (INIS)

Trott, K.R.; Gesellschaft fuer Strahlen- und Umweltforschung m.b.H., Neuherberg/Muenchen

1976-01-01

In most tumours there is a disproportion between the tumour cells and vascular connective tissue. A lack of oxygen depending on extent and duration, leads to changes of the metabolism and of the proliferative properties of the cells, to an increase of radiation resistance and to a reduction of the ability to recover from radiation injuries. Finally with longer duration, hypoxy leads to cell killing. As a result of irradiation, a reoxygenation of a part of the previous hypoxic tumour cell occurs more or less quickly. The time and topographic changes of these factors are involved in a complex manner in the radiotherapy of malignant tumours and essentially share the responsibility regarding the curative success of radiotherapy. (orig./LH) [de

Preferential expression and function of voltage-gated, O2-sensitive K+ channels in resistance pulmonary arteries explains regional heterogeneity in hypoxic pulmonary vasoconstriction: ionic diversity in smooth muscle cells.

Science.gov (United States)

Archer, Stephen L; Wu, Xi-Chen; Thébaud, Bernard; Nsair, Ali; Bonnet, Sebastien; Tyrrell, Ben; McMurtry, M Sean; Hashimoto, Kyoko; Harry, Gwyneth; Michelakis, Evangelos D

2004-08-06

Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (E(M)), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (I(K)) and a more hyperpolarized E(M) and were uniquely O2- and correolide-sensitive. The O2-sensitive current (active at -65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited I(K) 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide's effects. The hypoxia-sensitive, correolide-insensitive I(K) (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2- and correolide-sensitive I(K) like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.

Erythropoietin and a nonerythropoietic peptide analog promote aortic endothelial cell repair under hypoxic conditions: role of nitric oxide

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Heikal L

2016-08-01

Full Text Available Lamia Heikal,1 Pietro Ghezzi,1 Manuela Mengozzi,1 Blanka Stelmaszczuk,2 Martin Feelisch,2 Gordon AA Ferns1 1Brighton and Sussex Medical School, Falmer, Brighton, 2Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital and Institute for Life Sciences, Southampton, UK Abstract: The cytoprotective effects of erythropoietin (EPO and an EPO-related nonerythropoietic analog, pyroglutamate helix B surface peptide (pHBSP, were investigated in an in vitro model of bovine aortic endothelial cell injury under normoxic (21% O2 and hypoxic (1% O2 conditions. The potential molecular mechanisms of these effects were also explored. Using a model of endothelial injury (the scratch assay, we found that, under hypoxic conditions, EPO and pHBSP enhanced scratch closure by promoting cell migration and proliferation, but did not show any effect under normoxic conditions. Furthermore, EPO protected bovine aortic endothelial cells from staurosporine-induced apoptosis under hypoxic conditions. The priming effect of hypoxia was associated with stabilization of hypoxia inducible factor-1α, EPO receptor upregulation, and decreased Ser-1177 phosphorylation of endothelial nitric oxide synthase (NOS; the effect of hypoxia on the latter was rescued by EPO. Hypoxia was associated with a reduction in nitric oxide (NO production as assessed by its oxidation products, nitrite and nitrate, consistent with the oxygen requirement for endogenous production of NO by endothelial NOS. However, while EPO did not affect NO formation in normoxia, it markedly increased NO production, in a manner sensitive to NOS inhibition, under hypoxic conditions. These data are consistent with the notion that the tissue-protective actions of EPO-related cytokines in pathophysiological settings associated with poor oxygenation are mediated by NO. These findings may be particularly relevant to atherogenesis and postangioplasty restenosis. Keywords

In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions); Effets in vitro du piracetam sur la radiosensibilite des cellules hypoxiques (adapatation du test au MTT aux conditions d`hypoxie)

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Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T. [Universitaire Instelling Antwerpen, Antwerp (Belgium); Lagarde, P. [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Institut Bergonie, 33 - Bordeaux (France); Pooter, C.M.J. de [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Hopital de Middelheim, Anvers (Belgium); Chomy, F. [Institut Bergonie, 33 - Bordeaux (France)

1995-12-31

This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs.

Dose escalation of the hypoxic cell sensitizer etanidazole combined with ifosfamide, carboplatin, etoposide, and autologous hematopoietic stem cell support.

Science.gov (United States)

Elias, A D; Wheeler, C; Ayash, L J; Schwartz, G; Ibrahim, J; Mills, L; McCauley, M; Coleman, N; Warren, D; Schnipper, L; Antman, K H; Teicher, B A; Frei, E

1998-06-01

Multiple mechanisms of drug resistance contribute to treatment failure. Although high-dose therapy attempts to overwhelm these defenses pharmacologically, this approach is only successful in a fraction of treated patients. Many drug resistance mechanisms are shared between malignant and normal cells, but the expression of various drug resistance mechanisms associated with hypoxia is largely confined to tumor tissue. Thus, reversal of this mechanism is likely to provide a therapeutic advantage to the host. This study was designed to define the dose-limiting toxicities and maximum tolerated dose of etanidazole when it is given concurrently with high-dose ifosfamide, carboplatin, and etoposide (ICE), with hematopoietic stem cell support. The maximum tolerated doses of high-dose ICE were administered concurrently with dose escalations of etanidazole, a hypoxic cell sensitizer. All agents were given by 96-h continuous i.v. infusion beginning on day -7. Mesna uroprotection was provided. Autologous marrow and cytokine mobilized peripheral blood progenitor cells were reinfused on day 0. Granulocyte colony-stimulating factor was administered following reinfusion until the granulocytes recovered to > 1000/microliter. Fifty-five adults with advanced malignancies were enrolled in cohorts of five to nine patients. Four dose levels of etanidazole between 3 and 5.5 g/m2/day (12, 16, 20, and 22 g/m2 total doses) and two doses of carboplatin (1600 and 1800 mg/m2 total doses) were evaluated. Seven patients died of organ toxicity (13%); two each from veno-occlusive disease of liver and sepsis; and one each from sudden death, renal failure, and refractory thrombocytopenic hemorrhage. Five deaths occurred at the top dose level. One additional patient suffered a witnessed cardiorespiratory arrest from ventricular fibrillation and was resuscitated. Dose-dependent and largely reversible peripheral neuropathy was observed consisting of two syndromes: severe cramping myalgic/neuralgic pain

Cyclometalated Ruthenium(II) Anthraquinone Complexes Exhibit Strong Anticancer Activity in Hypoxic Tumor Cells.

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Zeng, Leli; Chen, Yu; Huang, Huaiyi; Wang, Jinquan; Zhao, Donglei; Ji, Liangnian; Chao, Hui

2015-10-19

Hypoxia is the critical feature of the tumor microenvironment that is known to lead to resistance to many chemotherapeutic drugs. Six novel ruthenium(II) anthraquinone complexes were designed and synthesized; they exhibit similar or superior cytotoxicity compared to cisplatin in hypoxic HeLa, A549, and multidrug-resistant (A549R) tumor cell lines. Their anticancer activities are related to their lipophilicity and cellular uptake; therefore, these physicochemical properties of the complexes can be changed by modifying the ligands to obtain better anticancer candidates. Complex 1, the most potent member of the series, is highly active against hypoxic HeLa cancer cells (IC50 =0.53 μM). This complex likely has 46-fold better activity than cisplatin (IC50 =24.62 μM) in HeLa cells. This complex tends to accumulate in the mitochondria and the nucleus of hypoxic HeLa cells. Further mechanistic studies show that complex 1 induced cell apoptosis during hypoxia through multiple pathways, including those of DNA damage, mitochondrial dysfunction, and the inhibition of DNA replication and HIF-1α expression, making it an outstanding candidate for further in vivo studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hypoxic cell radiosensitization by moderate hyperthermia and glucose deprivation

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Kim, J.H.; Kim, S.H.; Hahn, E.W.

1983-01-01

Cell culture studies were carried out to determine whether moderate hyperthermia reduces the oxygen enhancement ratio of cells under well-defined cultural conditions. Using asynchronously growing HeLa cells, the OER of cells with and without glucose was determined following exposure of cells to moderate hyperthermia, 40.5omicronC for 1 hr, immediately after X irradiation. The OER of cells with 5 mM glucose was 3.2, whereas the OER of glucose-deprived cells was reduced to 2.0. The pH of the cell culture medium was kept at 7.4 throughtout the experiments. The present finding may provide a clue toward further enhancing the radiosensitization of hypoxic cells by heat

Hypoxic cell radiosensitization by moderate hyperthermia and glucose deprivation

International Nuclear Information System (INIS)

Kim, J.H.; Kim, S.H.; Hahn, E.W.

1983-01-01

Cell culture studies were carried out to determine whether moderate hyperthermia reduces the oxygen enhancement ratio of cells under well-defined cultural conditions. Using asynchronously growing HeLa cells, the OER of cells with and without glucose was determined following exposure of cells to moderate hyperthermia, 40.5 degrees C for 1 hr, immediately after X irradiation. The OER of cells with 5 mM glucose was 3.2, whereas the OER of glucose-deprived cells was reduced to 2.0. The pH of the cell culture medium was kept at 7.4 throughout the experiments. The present finding may provide a clue toward further enhancing the radiosensitization of hypoxic cells by heat

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

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Schilling, Daniela; Bayer, Christine; Li, Wei; Molls, Michael; Vaupel, Peter; Multhoff, Gabriele

2012-01-01

Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1α (HIF-1α). Overexpression of HIF-1α is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy. Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1α levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1α levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1α upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1α levels and a concomitant increase in radiosensitivity. In summary, our data show a HIF-1α-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition.

Further characterization of 4-bromomisonidazole as a potential detector of hypoxic cells

International Nuclear Information System (INIS)

Rasey, J.S.; Krohn, K.A.; Grunbaum, Z.; Conroy, P.J.; Bauer, K.; Sutherland, R.M.

1985-01-01

[ 14 C]Bromomisonidazole was prepared by direct bromination of [ring-2] [ 14 C]misonidazole in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 μM [ 14 C]bromomisonidazole concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 μM[ 14 C]misonidazole may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with [ 14 C]misonidazole or [ 14 C]bromomisonidazole showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The data support the potential of radiolabled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent

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

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

2017-09-05

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

Studies of 99mTc-BnAO (HL-91): a non-nitroaromatic compound for hypoxic cell detection

International Nuclear Information System (INIS)

Zhang, X.; Melo, T.; Ballinger, J.R.; Rauth, A.M.

1998-01-01

Purpose: Solid tumours of similar type and stage can vary widely in their hypoxic cell fraction. Such cells may be prognostic for aggressive, metastatic, and radiation-resistant disease. A 99m technetium ( 99m Tc)-labelled non-nitroaromatic agent, butyleneamine oxime ( 99m Tc-BnAO) or HL-91 (Amersham International, Inc., Amersham, UK) has been evaluated both in vitro and in vivo for its possible efficacy as a noninvasive marker for the clinical detection of hypoxic cells in solid tumours. Materials and Methods: Suspension cultures of Chinese hamster ovary (CHO) cells under controlled levels of oxygen were used to measure the oxygen dependency of 99m Tc-BnAO accumulation. V79 cells grown as multilayers on a semipermeable membrane served as an in vitro model for drug penetration through the extravascular space of the tumour. C3H mice bearing KHT-C leg tumours were the in vivo models for selective drug accumulation as a function of time after i.v. administration of 99m Tc-BnAO. Results: 99m Tc accumulated selectively in hypoxic vs. aerobic cells, resulting in a 9 ± 2-fold differential in radioactivity per cell at 4 h. The k m for this selective accumulation was 20 ppm of oxygen. The labelled drug was equally effective in penetrating the cellular multilayer under aerobic or hypoxic conditions. In vivo measurements indicated favourable labelling of solid tumours containing hypoxic cells with 1% of the total activity per g of tumour, a tumour-to-blood ratio of 1.2, and a tumour-to-muscle ratio of 4.6 at 4 to 6 h after drug administration. In contrast to more lipophilic 99m Tc- labelled compounds, excretion was primarily via the urinary tract. Nitro-L-arginine selectively increased solid tumour labelling over normal tissue. Conclusions: 99m Tc-BnAO or HL-91 is a promising agent for clinical studies of tumour hypoxia, although the mechanism of its selective hypoxic cell accumulation remains unexplained

Cardiac biomarkers in neonatal hypoxic ischaemia.

LENUS (Irish Health Repository)

Sweetman, D

2012-04-01

Following a perinatal hypoxic-ischaemic insult, term infants commonly develop cardiovascular dysfunction. Troponin-T, troponin-I and brain natriuretic peptide are sensitive indicators of myocardial compromise. The long-term effects of cardiovascular dysfunction on neurodevelopmental outcome following perinatal hypoxic ischaemia remain controversial. Follow-up studies are warranted to ensure optimal cardiac function in adulthood. CONCLUSION: Cardiac biomarkers may improve the diagnosis of myocardial injury, help guide management, estimate mortality risk and may also aid in longterm neurodevelopmental outcome prediction following neonatal hypoxic-ischaemia.

Microenvironment around tumors and their radiation sensitivity. The possibility of molecular target for radiation sensitization

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Akimoto, Tetsuo; Ishikawa, Hitoshi [Gunma Univ., Maebashi (Japan). School of Medicine; Mitsuhashi, Norio [Tokyo Women' s Medical Coll. (Japan)

2001-12-01

There have been scarce studies concerning the effect of microenvironment around tumors on their radiation sensitivity and this review describes the influence of environmental factors of cell adhesion, growth factors, cytokines, hypoxia and angiogenesis on the sensitivity and response to radiation and on the signal transduction to consider the possibility of molecular target for radiation sensitization. Cell-cell adhesion and cell-matrix interaction in response to radiation may have a role in inducing apoptotic process like anti-apoptotic or pro-apoptotic one. Growth factors and cytokines can affect the tumor response to radiation in more extent than p53 gene status since apoptosis induction is not always an indication of radiation sensitivity in many tumors clinically encountered. Radiation sensitivity is low in tumor cells under hypoxic conditions and it is important to know the relationship between those hypoxic cell response and angiogenesis by factors like HIF (hypoxia-inducible factor)-1. Molecular targets for radiation sensitization are now under development and both basic and clinical studies are important for future application of those sensitizing agents for the radiotherapy of tumors. (K.H.)

Microenvironment around tumors and their radiation sensitivity. The possibility of molecular target for radiation sensitization

International Nuclear Information System (INIS)

Akimoto, Tetsuo; Ishikawa, Hitoshi

2001-01-01

There have been scarce studies concerning the effect of microenvironment around tumors on their radiation sensitivity and this review describes the influence of environmental factors of cell adhesion, growth factors, cytokines, hypoxia and angiogenesis on the sensitivity and response to radiation and on the signal transduction to consider the possibility of molecular target for radiation sensitization. Cell-cell adhesion and cell-matrix interaction in response to radiation may have a role in inducing apoptotic process like anti-apoptotic or pro-apoptotic one. Growth factors and cytokines can affect the tumor response to radiation in more extent than p53 gene status since apoptosis induction is not always an indication of radiation sensitivity in many tumors clinically encountered. Radiation sensitivity is low in tumor cells under hypoxic conditions and it is important to know the relationship between those hypoxic cell response and angiogenesis by factors like HIF (hypoxia-inducible factor)-1. Molecular targets for radiation sensitization are now under development and both basic and clinical studies are important for future application of those sensitizing agents for the radiotherapy of tumors. (K.H.)

Neuroprotective effects of ginsenoside Rg1-induced neural stem cell transplantation on hypoxic-ischemic encephalopathy

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Ying-bo Li

2015-01-01

Full Text Available Ginsenoside Rg1 is the major pharmacologically active component of ginseng, and is reported to have various therapeutic actions. To determine whether it induces the differentiation of neural stem cells, and whether neural stem cell transplantation after induction has therapeutic effects on hypoxic-ischemic encephalopathy, we cultured neural stem cells in 10-80 µM ginsenoside Rg1. Immunohistochemistry revealed that of the concentrations tested, 20 mM ginsenoside Rg1 had the greatest differentiation-inducing effect and was the concentration used for subsequent experiments. Whole-cell patch clamp showed that neural stem cells induced by 20 µM ginsenoside Rg1 were more mature than non-induced cells. We then established neonatal rat models of hypoxic-ischemic encephalopathy using the suture method, and ginsenoside Rg1-induced neural stem cells were transplanted via intracerebroventricular injection. These tests confirmed that neural stem cells induced by ginsenoside had fewer pathological lesions and had a significantly better behavioral capacity than model rats that received saline. Transplanted neural stem cells expressed neuron-specific enolase, and were mainly distributed in the hippocampus and cerebral cortex. The present data suggest that ginsenoside Rg1-induced neural stem cells can promote the partial recovery of complicated brain functions in models of hypoxic-ischemic encephalopathy.

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

International Nuclear Information System (INIS)

Ren Hongying; Cao Ying; Zhao, Qinjun; Li Jing; Zhou Cixiang; Liao Lianming; Jia Mingyue; Zhao Qian; Cai Huiguo; Han Zhongchao; Yang Renchi; Chen Guoqiang; Zhao, R.C.

2006-01-01

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O 2 , bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G 2 /S/M phase cells increased evidently under 8% O 2 condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O 2 condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl 2 ) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation

Intervention with the hypoxic tumor cell sensitizer etanidazole in the combined modality treatment of limited stage small-cell lung cancer. A one-institution study

International Nuclear Information System (INIS)

Urtasun, Raul C.; Palmer, Martin; Kinney, Brenda; Belch, Andrew; Hewitt, Joanne; Hanson, John

1998-01-01

Purpose: We report the toxicity, patterns of failure and survival of a cohort of patients with limited disease (LD) small-cell lung cancer (SCLC) treated with combined radiation and chemotherapy. During the course of thoracic irradiation, we added intravenous (i.v.) etanidazole (SR-2508, a third-generation 2-nitroimidazole) as a hypoxic cell sensitizer in an attempt to reduce the primary local failure rate and improve survival. Methods and Materials: Between July 1988 and August 1990, 30 consecutive patients with limited disease SCLC were enrolled and treated on a Phase II protocol receiving a standard combination chemotherapy regimen utilizing i.v. cisplatin 25 mg/m 2 /day x 3 days, i.v. etoposide 100 mg/m 2 /day x 3 days alternating with intravenous cyclophosphamide 1000 mg/m 2 /day, intravenous doxorubicin 15 mg/m 2 , and intravenous vincristine 2 mg (CAV) to a total of six cycles every 3 weeks. Radiotherapy and etanidazole were started after the first cycle of chemotherapy. Etanidazole was administered intravenously at a dose of 2 g/m 2 three times per week for a total of 30 g/m 2 during the course of thoracic radiation that delivered 50.00 Gy tumor dose in 25 fractions in an overall time of 6 weeks. Results: The overall response rate of the primary lesion in the thorax was 96% (CR + PR), with 64% complete responses. The median time to treatment failure was 18 months. Of the patients that have relapsed, only 18% failed in the thorax (alone or concomitant with other sites). This is a marked improvement compared to the 40-50% rate reported in the literature. The 2-year crude survival was 46%. The 3- and 5-year crude survival rate with no evidence of disease was 33 and 30%, respectively. We have observed a 10% increase in the incidence of transient etanidazole related peripheral neuropathies compared to previous etanidazole studies not utilizing systemic chemotherapy. There was no increased incidence of radiation esophagitis, pulmonary toxicity, or nephro- or

Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice.

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Lei Chen

Full Text Available Growing evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs enhance wound repair via paracrine. Because the extent of environmental oxygenation affects the innate characteristics of BM-MSCs, including their stemness and migration capacity, the current study set out to elucidate and compare the impact of normoxic and hypoxic cell-culture conditions on the expression and secretion of BM-MSC-derived paracrine molecules (e.g., cytokines, growth factors and chemokines that hypothetically contribute to cutaneous wound healing in vivo. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA analyses of normoxic and hypoxic BM-MSCs and their conditioned medium fractions showed that the stem cells expressed and secreted significantly higher amounts of basic fibroblast growth factor (bFGF,vascular endothelial growth factor A (VEGF-A interleukin 6 (IL-6 and interleukin 8 (IL-8 under hypoxic conditions. Moreover, hypoxic BM-MSC-derived conditioned medium (hypoCM vs. normoxic BM-MSC-derived conditioned medium (norCM or vehicle control medium significantly enhanced the proliferation of keratinocytes, fibroblasts and endothelial cells, the migration of keratinocytes, fibroblasts, endothelial cells and monocytes, and the formation of tubular structures by endothelial cells cultured on Matrigel matrix. Consistent with these in vitro results, skin wound contraction was significantly accelerated in Balb/c nude mice treated with topical hypoCM relative to norCM or the vehicle control. Notably increased in vivo cell proliferation, neovascularization as well as recruitment of inflammatory macrophages and evidently decreased collagen I, and collagen III were also found in the hypoCM-treated group. These findings suggest that BM-MSCs promote murine skin wound healing via hypoxia-enhanced paracrine.

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

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Frank Pajonk

2006-12-01

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

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

International Nuclear Information System (INIS)

Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

2012-01-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G 0 /G 1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G 0 /G 1 phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: ► miR-210 downregulation radiosensitized hypoxic hepatoma. ► AIFM3 was identified as a direct target gene of miR-210. ► miR-210 might be a therapeutic target to hypoxic hepatoma.

Peritoneal milky spots serve as a hypoxic niche and favor gastric cancer stem/progenitor cell peritoneal dissemination through hypoxia-inducible factor 1α.

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Miao, Zhi-Feng; Wang, Zhen-Ning; Zhao, Ting-Ting; Xu, Ying-Ying; Gao, Jian; Miao, Feng; Xu, Hui-Mian

2014-12-01

Peritoneal dissemination is the most common cause of death in gastric cancer patients. The hypoxic microenvironment plays a major role in controlling the tumor stem cell phenotype and is associated with patients' prognosis through hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor that responds to hypoxic stimuli. During the peritoneal dissemination process, gastric cancer stem/progenitor cells (GCSPCs) are thought to enter into and maintained in peritoneal milky spots (PMSs), which have hypoxic microenvironments. However, the mechanism through which the hypoxic environment of PMSs regulated GCSPC maintenance is still poorly understood. Here, we investigated whether hypoxic PMSs were an ideal cancer stem cell niche suitable for GCSPC engraftment. We also evaluated the mechanisms through which the HIF-1α-mediated hypoxic microenvironment regulated GCSPC fate. We observed a positive correlation between HIF-1α expression and gastric cancer peritoneal dissemination (GCPD) in gastric cancer patients. Furthermore, the GCSPC population expanded in primary gastric cancer cells under hypoxic condition in vitro, and hypoxic GCSPCs showed enhanced self-renewal ability, but reduced differentiation capacity, mediated by HIF-1α. In an animal model, GCSPCs preferentially resided in the hypoxic zone of PMSs; moreover, when the hypoxic microenvironment in PMSs was destroyed, GCPD was significantly alleviated. In conclusion, our results demonstrated that PMSs served as a hypoxic niche and favored GCSPCs peritoneal dissemination through HIF-1α both in vitro and in vivo. These results provided new insights into the GCPD process and may lead to advancements in the clinical treatment of gastric cancer. © 2014 The Authors. STEM CELLS Published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

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Hongying, Ren; Huiguo, Cai; Zhongchao, Han; Renchi, Yang; Zhao, Qinjun [State Key Lab of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin (China); Ying, Cao; Jing, Li [Institute of Basic Medical Sciences and School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Cixiang, Zhou [Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Science-SSMU, Shanghai (China); Lianming, Liao; Mingyue, Jia [Institute of Basic Medical Sciences and School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Qian, Zhao [Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Science-SSMU, Shanghai (China); Guoqiang, Chen [Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Science-SSMU, Shanghai (China); Zhao, R C [State Key Lab of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin (China); [Institute of Basic Medical Sciences and School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China)]. E-mail: chunhuaz@public.tpt.tj.cn

2006-08-18

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O{sub 2}, bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G{sub 2}/S/M phase cells increased evidently under 8% O{sub 2} condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O{sub 2} condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl{sub 2}) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.

TRAIL overexpression co-regulated by Egr1 and HRE enhances radiosensitivity of hypoxic A549 cells depending on its apoptosis inducing role.

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Yang, Yan-Ming; Fang, Fang; Li, Xin; Yu, Lei; Wang, Zhi-Cheng

2017-01-01

Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

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Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

2012-05-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

DEFF Research Database (Denmark)

Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

2011-01-01

Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

Contrasting hypoxic effects on breast cancer stem cell hierarchy is dependent on ER-α status.

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Harrison, Hannah; Rogerson, Lynsey; Gregson, Hannah J; Brennan, Keith R; Clarke, Robert B; Landberg, Göran

2013-02-15

Tumor hypoxia is often linked to decreased survival in patients with breast cancer and current therapeutic strategies aim to target the hypoxic response. One way in which this is done is by blocking hypoxia-induced angiogenesis. Antiangiogenic therapies show some therapeutic potential with increased disease-free survival, but these initial promising results are short lived and followed by tumor progression. We hypothesized that this may be due to altered cancer stem cell (CSC) activity resulting from increased tumor hypoxia. We studied the effects of hypoxia on CSC activity, using in vitro mammosphere and holoclone assays as well as in vivo limiting dilution experiments, in 13 patient-derived samples and four cell lines. There was a HIF-1α-dependent CSC increase in ER-α-positive cancers following hypoxic exposure, which was blocked by inhibition of estrogen and Notch signaling. A contrasting decrease in CSC was seen in ER-α-negative cancers. We next developed a xenograft model of cell lines and patient-derived samples to assess the hypoxic CSC response. Varying sizes of xenografts were collected and analyzed for HIF1-α expression and CSC. The same ER-α-dependent contrasting hypoxic-CSC response was seen validating the initial observation. These data suggest that ER-α-positive and negative breast cancer subtypes respond differently to hypoxia and, as a consequence, antiangiogenic therapies will not be suitable for both subgroups.

Tracking hypoxic signaling within encapsulated cell aggregates.

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Skiles, Matthew L; Sahai, Suchit; Blanchette, James O

2011-12-16

, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 10(10) pfu/mL. Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion. Western blot analysis of encapsulated cells in 20% and 1% oxygen also

Effects of radiation sensitivity of 17-DMAG on hypoxic carcinima cells

International Nuclear Information System (INIS)

Wu Xiaofen; Hong Chengjiao; Zhang Baoguo

2011-01-01

Objective: To investigate the radiosensitivity effect of 17-DMAG on human HeLa cells of cervical cancer, which was induced by Hypoxia mimetic cobalt chloride(CoCl 2 ). Methods: First, HeLa cells was treated by different concentrations of CoCl 2 for 24 hours. And HIF-1α protein level was detected by Western blot analysis. Then the HeLa cells was dealed with CoCl 2 in concentration of 200 μmol/L. Cells were pre-treated with different concentrations of 17-DMAG 16h after receiving different doses of 2, 4, 6, 8 Gy irradiation. The method of colony-forming was used to detect the radiation sensitivity. HIF-la protein level was detected by Western blot analysis. Results: When CoCl 2 was in concentration in 200 μmol/L, the HIF-1a protein expression was highest; HeLa cells were cultured in different concenreations of 17-DMAG and exposed to different doses of Irradiation, the cell servival rate was significant(P<0.01); After 17-DMAG pretreatment hypoxia of HeLa cells, HIF-1a protein expression with the drug concentration were inhibited significantly. Conclusions: 17-DMAG pretreatment hypoxia of HeLa cells can enhance radiosensitization and the mechanism may be through inhibition of HIF-1a protein expression. (authors)

[SOS response of DNA repair and genetic cell instability under hypoxic conditions].

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Vasil'eva, S V; Strel'tsova, D A

2011-01-01

The SOS DNA repair pathway is induced in E. coli as a multifunctional cell response to a wide variety of signals: UV, X or gamma-irradiation, mitomycin C or nalidixic acid treatment, thymine starvation, etc. Triggering of the system can be used as a general and early sign of DNA damage. Additionally, the SOS-response is known to be an "error-prone" DNA repair pathway and one of the sources of genetic instability. Hypoxic conditions are established to be the major factor of genetic instability as well. In this paper we for the first time studied the SOS DNA repair response under hypoxic conditions induced by the well known aerobic SOS-inducers. The SOS DNA repair response was examined as a reaction of E. coli PQ37 [sfiA::lacZ] cells to UVC, NO-donating agents and 4NQO. Here we provide evidence that those agents were able to induce the SOS DNA repair response in E. coli at anaerobic growth conditions. The process does not depend on the transcriptional activity of the universal protein of E. col anaerobic growth Fnr [4Fe-4S]2+ or can not be referred to as an indicator of genetic instability in hypoxic conditions.

Evaluation of nitrobenzimidazoles as hypoxic cell radiosensitizers

International Nuclear Information System (INIS)

Wright, J.; Frank, L.R.; Bush, D.; Harrison, G.H.

1983-01-01

Radiobiological and pharmacokinetic assays were performed to determine the potential of 2-nitrobenzimidazole (NBI) as a hypoxic cell radiosensitizing agent. As judged by comparing survival curve slopes of Serratia marcescens irradiated under aerated and hypoxic conditions, the NBI enhancement ratio (ER) at 2 mM concentration was 2.4 +- 0.2, compared with an oxygen enhancement ratio of 3.3 +- 0.3. 2,5-Dinitrobenzimidazole (DNBI) was investigated in vitro; its ER was 3.0 +- 0.3 at 4 mM concentration. Very poor tissue penetration of DNBI precluded further testing in vivo. Acute toxic signs appeared in C3H/HeJ mice following ip injection of NBI at 100 mg/kg. These would be partly attributable to the stress caused by the high pH of the injection vehicle. The LD 50 was estimated to be 125 to 150 mg/kg. Mammary adenocarcinoma tumors grown in the flanks of these mice exhibited maximum NBI levels at 5 min postinjection (ip). Peak tumor radiosensitization occurred in the interval between 5 and 10 min postinjection. The ER for tumor regrowth delay was 2.1 +- 0.3 following 50 mg/kg injected into mice 5 min before irradiation. Functional evaluation up to 40 days after treatment revealed no evidence of neurological deficit

Evaluation of nitrobenzimidazoles as hypoxic cell radiosensitizers

International Nuclear Information System (INIS)

Wright, J.; Frank, L.R.; Bush, D.; Harrison, G.H.

1983-01-01

Radiobiological and pharmacokinetic assays were performed to determine the potential of 2-nitrobenzimidazole (NBI) as a hypoxic cell radiosensitizing agent. As judged by comparing survival curve slopes of Serratia marcescens irradiated under aerated and hypoxic conditions, the NBI enhancement ratio (ER) at 2 mM concentration was 2.4 +/- 0.2, compared with an oxygen enhancement ratio of 3.3 +/- 0.3. 2,5-Dinitrobenzimidazole (DNBI) was investigated in vitro; its ER was 3.0 +/- 0.3 at 4 mM concentration. Very poor tissue penetration of DNBI precluded further testing in vivo. Acute toxic signs appeared in C3H/HeJ mice following ip injection of NBI at 100 mg/kg. These would be partly attributable to the stress caused by the high pH of the injection vehicle. The LD50 was estimated to be 125-150 mg/kg. Mammary adenocarcinoma tumors grown in the flanks of these mice exhibited maximum NBI levels at 5 min postinjection (ip). Peak tumor radiosensitization occurred in the interval between 5 and 10 min postinjection. The ER for tumor regrowth delay was 2.1 +/- 0.3 following 50 mg/kg injected into mice 5 min before irradiation. Functional evaluation up to 40 days after treatment revealed no evidence of neurological deficit

Oxygen-sensitive potassium channels in chemoreceptor cell physiology: making a virtue of necessity.

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Gonzalez, Constancio; Vaquero, Luis M; López-López, José Ramón; Pérez-García, M Teresa

2009-10-01

The characterization of the molecular mechanisms involved in low-oxygen chemotransduction has been an active field of research since the first description of an oxygen-sensitive K(+) channel in rabbit carotid body (CB) chemoreceptor cells. As a result, a large number of components of the transduction cascade, from O(2) sensors to O(2)-sensitive ion channels, have been found. Although the endpoints of the process are analogous, the heterogeneity of the elements involved in the different chemoreceptor tissues precludes a unifying theory of hypoxic signaling, and it has been a source of controversy. However, when these molecular constituents of the hypoxic cascade are brought back to their physiological context, it becomes clear that the diversity of mechanisms is necessary to build up an integrated cellular response that demands the concerted action of several O(2) sensors and several effectors.

Suicide of EMT-6 tumor cells by decays from radioactively-labelled sensitizer adducts

International Nuclear Information System (INIS)

Roa, W.H.Y.; Chapman, J.D.

1987-01-01

Nitroaromatic radiosensitizers become metabolically bound preferentially to hypoxic cells and at least 10/sup 9/ adducts/cell can be tolerated as non-toxic. EMT-6 tumor cells have been incubated in hypoxia in the presence of /sup 3/H-Misonidazole and /sup 125/I-Azomycin Riboside for various times and the amount of /sup 3/H or /sup 125/I bound/cell was determined. Cells were stored as monolayers at 25 0 C for up to 96 hr to accumulate radioactive decays and transferred at various times to 37 0 C for colony-forming assays. No radiation inactivation was measured in cells which had incorporated at least 10/sup 6/ /sup 3/H or 10/sup 5/ /sup 125/I atoms. Previous studies had shown that -- 1% of MISO adducts to EMT-6 cells was associated with cellular DNA. These data indicate that the radiation-induced damage produced by these quantities of bound /sup 3/H or /sup 125/I causes little or not cell inactivation. The results of current studies to measure the colony-forming ability of sensitizer-labelled cells which have been stored in liquid nitrogen to facilitate the accumulation of more decays will be reported. These data suggest that a ''sensitizer-adduct suicide technique'' as a hypoxic cell selective adjunct to other cancer therapies is not feasible. These data are also instructive for those who attempt to develop radiolabelled ''tumor specific'' antibodies for therapeutic purposes

Keynote address: hypoxic cell sensitizers: clinical developments

International Nuclear Information System (INIS)

Dische, S.

1989-01-01

Tumors having small islands of cells should be radiosensitive, and those having large masses, radioresistant. This was found to be the case and there is a ready explanation for this: the outside cells are close to blood vessels and will thus be well supplied with oxygen, while the inside cells are not well placed in this respect. Now it is known that cells well supplied with oxygen are radiosensitive, compared to the same cells deprived of oxygen; it is therefore likely that for this reason, variation in blood supply, that the outside cells are more easily destroyed than those within.''29 references

Hypoxic Preconditioning Promotes the Bioactivities of Mesenchymal Stem Cells via the HIF-1?-GRP78-Akt Axis

OpenAIRE

Lee, Jun Hee; Yoon, Yeo Min; Lee, Sang Hun

2017-01-01

Mesenchymal stem cells (MSC) are ideal materials for stem cell-based therapy. As MSCs reside in hypoxic microenvironments (low oxygen tension of 1% to 7%), several studies have focused on the beneficial effects of hypoxic preconditioning on MSC survival; however, the mechanisms underlying such effects remain unclear. This study aimed to uncover the potential mechanism involving 78-kDa glucose-regulated protein (GRP78) to explain the enhanced MSC bioactivity and survival in hindlimb ischemia. ...

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

International Nuclear Information System (INIS)

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

2003-01-01

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

Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

International Nuclear Information System (INIS)

Rutgers, D.H.; Niessen, D.P.P.; Linden, P.M. van der

1987-01-01

Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1,S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle. Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore FCM was used to monitor radiation induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (Tsub(c)) of approximately 84 hr, a subpopulation with a Tsub(c) of approximately 21 hr occurred. (author)

The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells

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Liang Dongming

2012-05-01

Full Text Available Abstract Background To study whether hypoxia influences the stem-like properties of ovarian cancer cells and their biological behavior under hypoxia. Method Ovarian cancer cell lines ES-2 and OVCAR-3 were cultivated in different oxygen tensions for proliferation, cell cycling and invasion analyses. The clonogenic potential of cells was examined by colony formation and sphere formation assays. Stem cell surface markers, SP and CD44bright and CD44dim cells were analyzed by flow cytometry. Protein expression of HIF-1α, HIF-2α, Ot3/4 and Sox2 were investigated by Western blotting. Results Both cell lines cultivated at hypoxic condition grew relatively slowly with extended G0/G1 phase. However, if the cells were pre-treated under 1% O2 for 48 hrs before brought back to normoxia, the cells showed significantly higher proliferation rate with higher infiltration capability, and significant more colonies and spheres, in comparison to the cells always cultivated under normoxia. CD44bright cells expressed significantly higher levels of Oct3/4 and Sox2 than the CD44dim cells and formed significantly more clones and spheres examined in vitro. Hypoxic treatment of the cells resulted in stronger CD44 expression in both cell lines, and stronger CD133 expression in the OVCAR-3 cell line. In parallel with these findings, significantly increased number of side population (SP cells and up-regulated expression of Oct3/4 and Sox2 in both ES-2 and OVCAR-3 cell lines were observed. Conclusion We conclude that ovarian cancer cells survive hypoxia by upgrading their stem-like properties through up-regulation of stemness-related factors and behave more aggressively when brought back to higher oxygen environment.

Induction of cancer cell death by proton beam in tumor hypoxic region

International Nuclear Information System (INIS)

Lee, Y. M.; Heo, T. R.; Lee, K. B.; Jang, K. H.; Kim, H. N.; Lee, S. H.; Jeong, M. H.

2008-04-01

Proton beam has been applied to treat various tumor patients in clinical studies. However, it is still undefined whether proton radiation can inhibit the blood vessel formation and induce the cell death in vascular endothelial cells in growing organs. The aim of this study are first, to develop an optimal animal model for the observation of blood vessel development with low dose of proton beam and second, to investigate the effect of low dose proton beam on the inhibition of blood vessel formation induced by hypoxic conditions. In this study, flk1-GFP transgenic zebrafish embryos were used to directly visualize and determine the inhibition of blood vessels by low dose (1, 2, 5 Gy) of proton beam with spread out Bragg peak (SOBP). And we observed cell death by acridine orange staining at 96 hours post fertilization (hpf) stage of embryos after proton irradiation. We also compared the effects of proton beam with those of gamma-ray. An antioxidant, N-acetyl cystein (NAC) was used to investigate whether reactive oxygen species (ROS) were involved in the cell deaths induced by proton irradiation. Irradiated flk-1-GFP transgenic embryos with proton beam irradiation (35 MeV, spread out Bragg peak, SOBP) demonstrated a marked inhibition of embryonic growth and an altered fluorescent blood vessel development in the trunk region. When the cells with DNA damage in the irradiated zebrafish were stained with acridine orange, green fluorescent cell death spots were increased in trunk regions compared to non-irradiated control embryos. Proton beam also significantly increased the cell death rate in human umbilical vein endothelial cells (HUVEC), but pretreatment of N-acetyl cystein (NAC), an antioxidant, recovered the proton-induced cell death rate (p<0.01). Moreover, pretreatment of NAC abrogated the effect of proton beam on the inhibition of trunk vessel development and malformation of trunk truncation. From this study, we found that proton radiation therapy can inhibit the

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

Science.gov (United States)

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

2016-07-01

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

c-Kit-positive cardiac stem cells nested in hypoxic niches are activated by stem cell factor reversing the aging myopathy.

Science.gov (United States)

Sanada, Fumihiro; Kim, Junghyun; Czarna, Anna; Chan, Noel Yan-Ki; Signore, Sergio; Ogórek, Barbara; Isobe, Kazuya; Wybieralska, Ewa; Borghetti, Giulia; Pesapane, Ada; Sorrentino, Andrea; Mangano, Emily; Cappetta, Donato; Mangiaracina, Chiara; Ricciardi, Mario; Cimini, Maria; Ifedigbo, Emeka; Perrella, Mark A; Goichberg, Polina; Choi, Augustine M; Kajstura, Jan; Hosoda, Toru; Rota, Marcello; Anversa, Piero; Leri, Annarosa

2014-01-03

Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.

Effects of 5 Thio-D-Glucose on cellular adenosine triphosphate levels and deoxyribonucleic acid rejoining in hypoxic and aerobic Chinese hamster cells

International Nuclear Information System (INIS)

Nagle, W.A.; Moss, A.J. Jr.; Roberts, H.G. Jr.; Baker, M.L.

1980-01-01

Intracellular adenosine triphosphate (ATP) levels were measured in both hypoxic and aerobic cultures of V79 Chinese hamster cells treated with 5-thio-D-glucose (5-SH-D-Glc). This glucose analog, a known inhibitor of D-glucose transport and metabolism, reduced ATP in cell cultures allowed to become hypoxic by cell metabolism, but not in aerobic cultures treated similarly. Cells depleted of ATP were unable to rejoin x-ray induced deoxyribonucleic acid (DNA) strand breaks as measured by the alkaline sucrose gradient sedimentation technique. The inference for radiation therapy is that inhibition of glucose metabolism selectively depletes energy reserves in hypoxic cells, rendering these cells more radiosensitive and leading to a more effective tumor treatment

Fetal stress and programming of hypoxic/ischemic-sensitive phenotype in the neonatal brain: mechanisms and possible interventions.

Science.gov (United States)

Li, Yong; Gonzalez, Pablo; Zhang, Lubo

2012-08-01

Growing evidence of epidemiological, clinical and experimental studies has clearly shown a close link between adverse in utero environment and the increased risk of neurological, psychological and psychiatric disorders in later life. Fetal stresses, such as hypoxia, malnutrition, and fetal exposure to nicotine, alcohol, cocaine and glucocorticoids may directly or indirectly act at cellular and molecular levels to alter the brain development and result in programming of heightened brain vulnerability to hypoxic-ischemic encephalopathy and the development of neurological diseases in the postnatal life. The underlying mechanisms are not well understood. However, glucocorticoids may play a crucial role in epigenetic programming of neurological disorders of fetal origins. This review summarizes the recent studies about the effects of fetal stress on the abnormal brain development, focusing on the cellular, molecular and epigenetic mechanisms and highlighting the central effects of glucocorticoids on programming of hypoxic-ischemic-sensitive phenotype in the neonatal brain, which may enhance the understanding of brain pathophysiology resulting from fetal stress and help explore potential targets of timely diagnosis, prevention and intervention in neonatal hypoxic-ischemic encephalopathy and other brain disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

Release from the Crabtree effect by hypoxic cell radiosensitizers

International Nuclear Information System (INIS)

Mustea, I.; Bara, A.

1979-01-01

The Crabtree effect can be observed when the 0 2 consumption of tumour cells or of mammalian cells grown in culture is measured in physiological medium containing glucose. The effect of 2 hypoxic cell radiosensitizers, misonidazole and NDPP, on the 0 2 consumption of Ehrlich ascites tumour cells was compared in media with and without glucose. A stimulatory effect on 0 2 consumption was found for 5-20 mM misonidazole as well as for 0.5mM NDPP, both in media containing 10 -2 M glucose. Thus glucose induced a Crabtree effect in Ehrlich tumour cells, expressed as 38-45% inhibition of 0 2 consumption relative to that in the same medium without glucose. The stimulatory effect of misonidazole and NDPP on 0 2 utilization in medium with glucose undoubtedly appeared as a release from the Crabtree effect. (author)

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction

International Nuclear Information System (INIS)

Rouschop, Kasper M.A.; Dubois, Ludwig; Schaaf, Marco B.E.; Beucken, Twan van den; Lieuwes, Natasja; Keulers, Tom G.H.; Savelkouls, Kim G.M.; Bussink, Johan; Kogel, Albert J. van der; Koritzinsky, Marianne; Wouters, Bradly G.

2011-01-01

Background and purpose: Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation. Material and methods: The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells. Results: We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation. Conclusions: Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.

Hypoxic hypoxia as a means of modifying radiosensibility

International Nuclear Information System (INIS)

Neumeister, K.; Niemiec, C.; Bolck, M.; Jahns, J.; Kamprad, F.; Arnold, P.; Johannsen, U.; Koch, F.; Mehlhorn, G.

1977-01-01

Following an overview of the various possibilities of creating hypoxia in mammals, the problem of reducing radioresistance of hypoxic tumor cells is treated. Furthermore, the results of irradiation experiments with mice, rats and pigs breathing hypoxic mixtures of O 2 and N 2 are given and discussed with a view to applying hypoxic hypoxia in the radiotherapy of human tumors. (author)

Considerations on hypoxic conditions. On the past setback of classic radiation biology

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Nakatsugawa, Shigekazu; Klimova, S.V.; Tamasu, Shogo; Nakamura, Hideaki; Murayama, Chieko

2002-01-01

Considerations on hypoxic cancer cell environment are made on classic radiation biology concept and on a new proposal of the anti-cancer strategy. Classic radiation biology knowledge of hypoxic cancer cells has produced many of clinical trials, which, however, have failed after all. This is because the knowledge is that the cells are recognized to be in a rather static hypoxic condition. Based on authors' investigations, made is the proposal that improvement of dynamic, acute hypoxic conditions yielded via blood circulation between the heterogeneous malignant cancer cells and the dynamic homeostatic systems of normal cells including immunity is important as one of cancer therapy approaches. (N.I.)

Synthesis of a fluorine-18 labeled hypoxic cell sensitizer

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Jerabek, P.A.; Dischino, D.D.; Kilbourn, M.R.; Welch, M.J.

1984-01-01

The objective of this work was to synthesize a positron emitting radiosensitizing agent as a potential in vivo marker of hypoxic regions within tumors, and ischemic areas of the heart and brain. The method involved radiochemical synthesis of fluorine-18 labeled 1-(2-nitro-imidazolyl)-3-fluoro-2-propanol via nucleophilic ring opening of 1-(2,3-epoxypropyl)2-nitro-imidzole by fluorine-18 labeled tetrabutylammonium fluoride (TBAF). Fluroine-18 TBAF was prepared by the exchange reaction of TBAF with aqueous flourine-18 produced by proton bombardment of enriched oxygen-18 water. The aqueous solution was evaporated carefully by azeotropic distillation with acetonitrile. The fluorine-18 labeled TBAF was taken up in N,N-dimethylacetamide or dimethysulfoxide, then reacted with the episode at 60C for 30 minutes. Separation and identification of the fluorine-18 labeled products by high performance liquid chromatography showed a radioactive peak with a retention time identical to that of 1-(2-nitro-1-imidazolyl)-3-fluoro-2-propanol and a second radioactive peak with a retention time three minutes longer in addition to unreacted fluorine-18 labeled TBAF. The second radioactive peak may represent fluorine-18 labeled 1-2-nitro-1-imidazolyl)-2-fluoro-3-propanol. The average radiochemical yield from reactions run in N,N-dimethylacetamide using 20 micromoles of TBAF and 1-2 mg of the epoxide was l7% in a synthesis time of about 40 minutes. The synthesis of fluorohydrins by the reaction of fluorine-18 labeled TBAF on epoxides represents a new method for the preparation of fluorine-18 labeled fluorohydrins

Gold nanoparticle cellular uptake, toxicity and radiosensitisation in hypoxic conditions

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Jain, Suneil; Coulter, Jonathan A.; Butterworth, Karl T.; Hounsell, Alan R.; McMahon, Stephen J.; Hyland, Wendy B.; Muir, Mark F.; Dickson, Glenn R.; Prise, Kevin M.; Currell, Fred J.; Hirst, David G.; O’Sullivan, Joe M.

2014-01-01

Background and purpose: Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored. Materials and methods: GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions. Results: GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred. Conclusions: GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy

Danshensu prevents hypoxic pulmonary hypertension in rats by inhibiting the proliferation of pulmonary artery smooth muscle cells via TGF-β-smad3-associated pathway.

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Zhang, Ning; Dong, Mingqing; Luo, Ying; Zhao, Feng; Li, Yongjun

2018-02-05

Hypoxic pulmonary hypertension is characterized by the remodeling of pulmonary artery. Previously we showed that tanshinone IIA, one lipid-soluble component from the Chinese herb Danshen, ameliorated hypoxic pulmonary hypertension by inhibiting pulmonary artery remodeling. Here we explored the effects of danshensu, one water-soluble component of Danshen, on hypoxic pulmonary hypertension and its mechanism. Rats were exposed to hypobaric hypoxia for 4 weeks to develop hypoxic pulmonary hypertension along with administration of danshensu. Hemodynamics and pulmonary arterial remodeling index were measured. The effects of danshensu on the proliferation of primary pulmonary artery smooth muscle cells and transforming growth factor-β-smad3 pathway were assessed in vitro. Danshensu significantly decreased the right ventricle systolic pressure, the right ventricle hypertrophy and pulmonary vascular remodeling index in hypoxic pulmonary hypertension rats. Danshensu also reduced the increased expression of transforming growth factor-β and phosphorylation of smad3 in pulmonary arteries in hypoxic pulmonary hypertension rats. In vitro, danshensu inhibited the hypoxia- or transforming growth factor-β-induced proliferation of primary pulmonary artery smooth muscle cells. Moreover, danshensu decreased the hypoxia-induced expression and secretion of transforming growth factor in primary pulmonary adventitial fibroblasts and NR8383 cell line, inhibited the hypoxia or transforming growth factor-β-induced phosphorylation of smad3 in rat primary pulmonary artery smooth muscle cells. These results demonstrate that danshensu ameliorates hypoxic pulmonary hypertension in rats by inhibiting the hypoxia-induced proliferation of pulmonary artery smooth muscle cells, and the inhibition effects is associated with transforming growth factor-β-smad3 pathway. Therefore danshensu may be a potential treatment for hypoxic pulmonary hypertension. Copyright © 2017 Elsevier B.V. All rights

Hypoxic Preconditioning Promotes the Bioactivities of Mesenchymal Stem Cells via the HIF-1α-GRP78-Akt Axis.

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Lee, Jun Hee; Yoon, Yeo Min; Lee, Sang Hun

2017-06-21

Mesenchymal stem cells (MSC) are ideal materials for stem cell-based therapy. As MSCs reside in hypoxic microenvironments (low oxygen tension of 1% to 7%), several studies have focused on the beneficial effects of hypoxic preconditioning on MSC survival; however, the mechanisms underlying such effects remain unclear. This study aimed to uncover the potential mechanism involving 78-kDa glucose-regulated protein (GRP78) to explain the enhanced MSC bioactivity and survival in hindlimb ischemia. Under hypoxia (2% O₂), the expression of GRP78 was significantly increased via hypoxia-inducible factor (HIF)-1α. Hypoxia-induced GRP78 promoted the proliferation and migration potential of MSCs through the HIF-1α-GRP78-Akt signal axis. In a murine hind-limb ischemia model, hypoxic preconditioning enhanced the survival and proliferation of transplanted MSCs through suppression of the cell death signal pathway and augmentation of angiogenic cytokine secretion. These effects were regulated by GRP78. Our findings indicate that hypoxic preconditioning promotes survival, proliferation, and angiogenic cytokine secretion of MSCs via the HIF-1α-GRP78-Akt signal pathway, suggesting that hypoxia-preconditioned MSCs might provide a therapeutic strategy for MSC-based therapies and that GRP78 represents a potential target for the development of functional MSCs.

Cell inactivation studies on yeast cells under euoxic and hypoxic condition using electron beam from microtron accelerator

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Praveen Joseph; Santhosh Acharya; Ganesh Sanjeev; Narayana, Y.; Bhat, N.N.

2011-01-01

In the case of sparsely ionizing radiation such as electron, the dose rate and the pattern of energy deposition of the radiation are the important physical factors which can affect the amount of damage in living cells. In the present study, the differences in the cell survival efficiency and dose rate effect in diploid yeast strains Saccharomyces cerevisiae X2180 and Saccharomyces cerevisiae D7 under euoxic and hypoxic condition have been quantified. Irradiation was carried out using 8 MeV pulsed electron beam from Microtron accelerator. The dose per pulse and pulse width of the beam used was 0.6 Gy and 2.3 μs, respectively, which correspond to an instantaneous dose rate of 2.6 x 10 5 Gy s -1 . For survival studies doses were delivered at a rate of 50 pulses per second (an average dose rate of 1,800 Gy s -1 ). Fricke and alanine dosimeters were used to measure the dose delivered to the sample. A significant difference in the dose response has been observed under euoxic and hypoxic condition. Dose rate effect has been studied by changing the pulse repetition rate of the Microtron and the dose rate used was from 180 to 1800 Gy min -1 . A significant dose rate effect was observed under euoxic condition for Saccharomyces cerevisiae X2180 but the same was absent under hypoxic condition. The dose rate effect was absent for Saccharomyces cerevisiae D7 under both irradiation condition. The survival curves are found to be sigmoidal in shape under both condition but with a wider shoulder under hypoxic condition. The D 0 value and the Oxygen Enhancement Ratio (OER) at that point have been derived. (author)

Coumestrol suppresses hypoxia inducible factor 1α by inhibiting ROS mediated sphingosine kinase 1 in hypoxic PC-3 prostate cancer cells.

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Cho, Sung-Yun; Cho, Sunmi; Park, Eunkyung; Kim, Bonglee; Sohn, Eun Jung; Oh, Bumsuk; Lee, Eun-Ok; Lee, Hyo-Jeong; Kim, Sung-Hoon

2014-06-01

Among many signals to regulate hypoxia inducible factor 1α (HIF-1α), sphingosine kinase 1 (SPHK1) is also involved in various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, molecular mechanisms of coumestrol were investigated on the SPHK1 and HIF-1α signaling pathway in hypoxic PC-3 prostate cancer cells. Coumestrol significantly suppressed SPHK1 activity and accumulation of HIF-1α in a time- and concentration-dependent manner in hypoxic PC-3 cells. In addition, coumestrol inhibited the phosphorylation status of AKT and glycogen synthase kinase-3β (GSK 3β) signaling involved in cancer metabolism. Furthermore, SPHK1 siRNA transfection, sphigosine kinase inhibitor (SKI), reactive oxygen species (ROS) enhanced the inhibitory effect of coumestrol on the accumulation of HIF-1α and the expression of pAKT and pGSK 3β in hypoxic PC-3 cells by combination index. Overall, our findings suggest that coumestrol suppresses the accumulation of HIF-1α via suppression of SPHK1 pathway in hypoxic PC-3 cells. Copyright © 2014. Published by Elsevier Ltd.

Transcriptional Profiling of Hypoxic Neural Stem Cells Identifies Calcineurin-NFATc4 Signaling as a Major Regulator of Neural Stem Cell Biology

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Moreno, Marta; Fernández, Virginia; Monllau, Josep M.; Borrell, Víctor; Lerin, Carles; de la Iglesia, Núria

2015-01-01

Summary Neural stem cells (NSCs) reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors (TFs) that regulate NSC biology under physiologic hypoxia are poorly understood. Here we have performed gene set enrichment analysis (GSEA) of microarray datasets from hypoxic versus normoxic NSCs with the aim of identifying pathways and TFs that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. Integration of TF target (TFT) and pathway enrichment analysis identified the calcium-regulated TF NFATc4 as a major candidate to regulate hypoxic NSC functions. Nfatc4 expression was coordinately upregulated by top hypoxia-activated TFs, while NFATc4 target genes were enriched in hypoxic NSCs. Loss-of-function analyses further revealed that the calcineurin-NFATc4 signaling axis acts as a major regulator of NSC self-renewal and proliferation in vitro and in vivo by promoting the expression of TFs, including Id2, that contribute to the maintenance of the NSC state. PMID:26235896

Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

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Pilgaard, L.; Lund, P.; Duroux, M.; Lockstone, H.; Taylor, J.; Emmersen, J.; Fink, T.; Ragoussis, J.; Zachar, V.

2009-01-01

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

International Nuclear Information System (INIS)

Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra; De Ridder, Mark

2013-01-01

Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes

Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

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Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium); De Ridder, Mark, E-mail: mark.deridder@uzbrussel.be [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium)

2013-03-01

Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.

Stem Cell Therapy for Neonatal Hypoxic-Ischemic Encephalopathy

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Gabriel eGonzales-Portillo

2014-08-01

Full Text Available Treatments for neonatal hypoxic ischemic encephalopathy (HIE have been limited. The aim of this paper is to offer translational research guidance on stem cell therapy for neonatal HIE by examining clinically relevant animal models, practical stem cell sources, safety and efficacy of endpoint assays, as well as a general understanding of modes of action of this cellular therapy. In order to do so, we discuss the clinical manifestations of HIE, highlighting its overlapping pathologies with stroke providing insights on the potential of cell therapy, currently investigated in stroke, for HIE. To this end, we draw guidance from recommendations outlined in Stem cell Therapeutics as an Emerging Paradigm for Stroke or STEPS, which have been recently modified to Baby STEPS to cater for the neonatal symptoms of HIE. These guidelines recognized that neonatal HIE exhibits distinct disease symptoms from adult stroke in need of an innovative translational approach that facilitates the entry of cell therapy in the clinic. Finally, new information about recent clinical trials, and insights into combination therapy are provided with the vision that stem cell therapy may benefit from available treatments, such as hypothermia, already being tested in children diagnosed with HIE.

Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton’s Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition

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Patcharee Prasajak

2013-01-01

Full Text Available The shortage of donor livers and hepatocytes is a major limitation of liver transplantation. Thus, generation of hepatocyte-like cells may provide alternative choice for therapeutic applications. In this study, we developed a new method to establish hepatocytes from Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs cell lines named WJMSCs-SUT1 and WJMSCs-SUT2 under hypoxic condition. This new method could rapidly drive both WJ-MSCs cell lines into hepatic lineage within 18 days. The achievement of hepatogenic differentiation was confirmed by the characterization of both phenotypes and functions. More than 80% MSCs-derived hepatocyte-like cells (MSCDHCs achieved functional hepatocytes including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. This study highlights the establishment of new hepatogenic induction protocol under hypoxic condition in order to mimic hypoxic microenvironment in typical cell physiology. In conclusion, we present a simple, high-efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs in hypoxic condition. The achievement of this method may overcome the limitation of donor hepatocytes and provides a new avenue for therapeutic value in cell-based therapy for life-threatening liver diseases, regenerative medicine, toxicity testing for pharmacological drug screening, and other medical related applications.

Absence of sensitization of reversions in yeast by metronidazole

International Nuclear Information System (INIS)

Krishnan, D.; Singh, D.R.; Mahajan, J.M.; Madhvanath, U.

1977-01-01

Metronidazole (Flagyl; 2-methyl-5-nitroimidazole-1-ethanol) is of potential clinical application as a radiosensitizer. Studies have been made of the effect of 1mM metronidazole on γ-induced heteroallelic recombination (reversion) under euoxic or hypoxic conditions in yeast strain BZ34, and compared with the effect on survival. The o.e.r. for reversions was 2.2, and very close to that for survival (2.3), indicating that oxygen equally sensitized gross cellular events such as killing, and genetic phenomena like reversions. The introduction of metronidazole during irradiation sensitized cells with killing as the end point only in hypoxic condition, whereas with reversion as the end point no sensitization due to the chemical was observed either in euoxic or hypoxic conditions. Since DNA damage is responsible for both cell-killing and mutations, the sensitizer might have been expected to have had an equal effect on both these end-points. The results show that only in a limited sense can metronidazole be considered to be an oxygen mimic. (U.K.)

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

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Orue, Andrea; Chavez, Valery; Strasberg-Rieber, Mary; Rieber, Manuel

2016-11-18

The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA. In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine. This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Hypoxic fraction and binding of misonidazole in EMT6/Ed multicellular tumor spheroids

International Nuclear Information System (INIS)

Franko, A.J.

1985-01-01

Misonidazole has been shown to bind selectively to hypoxic cells in tissue culture and to cells which are presumed to be chronically hypoxic in EMT6 spheroids and tumors. Thus it has considerable potential as a marker of hypoxic cells in vivo. To further evaluate this potential EMT6/Ed spheroids were used to quantitate misonidazole binding under conditions which resulted in hypoxic fractions between 0 and 1. The patterns of binding of 14 C-labeled misonidazole determined by autoradiography were consistent with the regions of radiobiological hypoxia as predicted by oxygen diffusion theory. The overall uptake of 3 H-labeled misonidazole by spheroids correlated well with the hypoxic fraction, although binding to aerobic cells and necrotic tissue contributed appreciably to the total label in the spheroids. It is concluded that misonidazole is an excellent marker of hypoxia in EMT6/Ed spheroids at the microscopic level, and the total amount bound per spheroid provides a potentially useful measure of the hypoxic fraction

The response of hypoxic cells in SCCVII murine tumors to treatment with cisplatin and x rays

International Nuclear Information System (INIS)

Yan, R.D.; Durand, R.E.

1991-01-01

Possible mechanisms of enhancement of radiation effects by cisplatin, including radiosensitization of hypoxic cells, drug-induced tumor reoxygenation, and inhibition of repair of sublethal radiation damage, were examined in the murine SCCVII model. Combination radiation/drug treatments were most effective when drug exposure preceded irradiation of animals breathing a reduced oxygen atmosphere, indicating that the primary interaction between the modalities was a cisplatin-induced increase in the oxygenation status of the acutely hypoxic cells in those tumors. Delivering cisplatin prior to or immediately after the first of two 5 Gy fractions was more effective than combinations with a single x-ray exposure, suggesting that proper sequences of the combined modalities may augment natural reoxygenation processes

The current status of drug development of hypoxic cell radiosensitizers and their potential role in gynecologic oncology

International Nuclear Information System (INIS)

Coleman, C.N.; Ballon, S.C.; Howes, A.E.; Martinez, A.; Halsey, J.; Hirst, V.K.

1984-01-01

Both laboratory and clinical data suggest that hypoxia contributes to the failure of radiotherapy to achieve local control of bulky gynecologic tumors. As part of a Phase I trial of hypoxic cell radiosensitizers, 19 women at Stanford University with advanced (n . 6) or recurrent (n . 13) pelvic neoplasms were treated with radiotherapy plus desmethylmisonidazole. Complete or partial response occurred in 42% of patients with some patients achieving local control for over 1 year. It is unknown if the sensitizer added to the results of radiotherapy alone. A Phase I trial of a theoretically superior sensitizer, SR-2508, is soon to begin. It is anticipated that the dose-limiting neurotoxicity seen with misonidazole and desmethylmisonidazole will either be eliminated or will occur at a much higher total dose of drug. Many patients with gynecologic tumors could potentially benefit from participation in the new drug trials

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Hypoxia-inducible factor-1α and Wnt/β-catenin signaling pathways promote the invasion of hypoxic gastric cancer cells.

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Liu, Hong-Lan; Liu, Dang; Ding, Guang-Rong; Liao, Peng-Fei; Zhang, Jun-Wen

2015-09-01

The present study aimed to examine the association between hypoxia-inducible factor (HIF)-1α and the Wnt/β-catenin signaling pathway in a hypoxic environment. The study also aimed to explore the possible mechanisms underlying the invasion of hypoxic gastric cancer cells in vitro and in vivo. The pcDNA™ 6.2‑GW/EmGFP‑miR‑β‑catenin plasmid was transfected into SGC‑7901 gastric cancer cells, resulting in cells with stable suppression of β‑catenin expression. The biological characteristics of the control, liposome, negative control, β‑catenin knockdown, hypoxia and hypoxia β‑catenin knockdown groups were tested using an invasion assay. The differences in the invasive capacity of the control, negative control and liposome groups were not statistically significant. However, the hypoxia group demonstrated a significantly enhanced invasive capacity, as compared with that in the control group (Phypoxic and control cells was high alongside increased HIF‑1α, β‑catenin, uPA and MMP‑7 levels according to western blot and immunohistochemical analyses, while growth and protein levels of tumors from hypoxic β‑catenin knockdown cells were significantly lower and those of β‑catenin knockdown cells were lowest. In conclusion, these results suggested that HIF‑1α activation was able to regulate the Wnt/β‑catenin pathway, and that HIF‑1α may be controlled by the Wnt/β‑catenin pathway. A potential mechanism underlying SGC‑7901 tumorigenicity is the activation of the Wnt/β‑catenin signaling pathway, which activates uPA and MMP‑7 expression and contributes to the enhanced invasion of hypoxic cancer cells.

Evaluation of 2-amino-5-nitrothiazole as a hypoxic cell radiosensitizer

International Nuclear Information System (INIS)

Rockwell, S.; Mroczkowski, Z.; Rupp, W.D.

1982-01-01

The nitroheterocyclic compound 2-amino-5-nitrothiazole (ANT) was evaluated as a hypoxic radiosensitizer. Experiments with bacteria showed that this agent was similar to misonidozole in radiosensitizing activity, but was less cytotoxic and less mutagenic than misonidazole. Experiments with EMT6 tumor cells in culture showed ANT to be an effective hypoxic radiosensitizer, although slightly less active than misonidazole, and to be less cytotoxic than misonidazole. ANT was more toxic to mice than misonidazole and produced a spectrum of symptoms, including hyperactivity and agitation, different from those of misonidazole. The toxicities of ANT and misonidazole were additive. The maximum levels of ANT achieveable in the tumors after ip injection of nontoxic doses of drug were low ( -4 M) and the radiosensitization obtainable with the drug in vivo was inferior to that obtainable with misonidazole. These findings suggest that nitrothiazoles might be an interesting class of nitroheterocyclic radiosensitizers, but that molecules with increased solubility and improved pharmacokinetics would be necessary for efficacy in vivo

A scenario and forecast model for Gulf of Mexico hypoxic area and volume

Science.gov (United States)

Scavia, Donald; Evans, Mary Anne; Obenour, Daniel R.

2013-01-01

For almost three decades, the relative size of the hypoxic region on the Louisiana-Texas continental shelf has drawn scientific and policy attention. During that time, both simple and complex models have been used to explore hypoxia dynamics and to provide management guidance relating the size of the hypoxic zone to key drivers. Throughout much of that development, analyses had to accommodate an apparent change in hypoxic sensitivity to loads and often cull observations due to anomalous meteorological conditions. Here, we describe an adaptation of our earlier, simple biophysical model, calibrated to revised hypoxic area estimates and new hypoxic volume estimates through Bayesian estimation. This application eliminates the need to cull observations and provides revised hypoxic extent estimates with uncertainties, corresponding to different nutrient loading reduction scenarios. We compare guidance from this model application, suggesting an approximately 62% nutrient loading reduction is required to reduce Gulf hypoxia to the Action Plan goal of 5,000 km2, to that of previous applications. In addition, we describe for the first time, the corresponding response of hypoxic volume. We also analyze model results to test for increasing system sensitivity to hypoxia formation, but find no strong evidence of such change.

Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

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Sung-Yun Cho

2013-01-01

Full Text Available Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

Ginsenoside Rg3 enhances radiosensitization of hypoxic oesophageal cancer cell lines through vascular endothelial growth factor and hypoxia inducible factor 1α.

Science.gov (United States)

Ge, Xiaolin; Zhen, Fuxi; Yang, Baixia; Yang, Xi; Cai, Jing; Zhang, Chi; Zhang, Sheng; Cao, Yuandong; Ma, Jianxin; Cheng, Hongyan; Sun, Xinchen

2014-06-01

To determine if the pretreatment of hypoxic human oesophageal carcinoma cell lines (EC109, TE1 and KYSE170) with ginsenoside Rg3 (Rg3) increases their radiosensitivity to X-rays. The growth inhibitory effect of different Rg3 concentrations was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Radiation sensitivity was measured using a clone formation assay and flow cytometry was used to measure the effects of Rg3 on radiation-induced apoptosis. Western blot analysis was used to measure the effects of Rg3 on the levels of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Rg3 inhibited EC109, TE1 and KYSE170 cell growth in a dose- and time-dependent manner. Pretreatment with 10 µmol/ml Rg3 increased EC109, TE1 and KYSE170 radiosensitivity. Rg3 plus radiation significantly increased the apoptosis rate compared with radiation alone. Rg3 also decreased VEGF and HIF-1α protein levels in EC109 cells in a dose-dependent manner. The combination of Rg3 and radiation increased the fragmentation of double-stranded DNA. Rg3 enhanced the radiosensitivity of human oesophageal carcinoma cell lines cultured under hypoxic conditions possibly by downregulating VEGF and HIF-1α protein levels. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Algorithm development and simulation outcomes for hypoxic head and neck cancer radiotherapy using a Monte Carlo cell division model

International Nuclear Information System (INIS)

Harriss, W.M.; Bezak, E.; Yeoh, E.

2010-01-01

Full text: A temporal Monte Carlo tumour model, 'Hyp-RT'. sim ulating hypoxic head and neck cancer has been updated and extended to model radiothcrapy. The aim is to providc a convenient radiobio logical tool for clinicians to evaluate radiotherapy treatment schedules based on many individual tumour properties including oxygenation. FORTRAN95 and JA YA havc been utilised to develop the efficient algorithm, which can propagate 108 cells. Epithelial cell kill is affected by dose, oxygenation and proliferativc status. Accelerated repopulation (AR) has been modelled by increasing the symmetrical stem cell division probability, and reoxygenation (ROx) has been modelled using random incremental boosts of oxygen to the cell po ulation throughout therapy. Results The stem cell percentage and the degree of hypoxia dominate tumour growth rate. For conventional radiotherapy. 15-25% more dose was required for a hypox ic versus oxic tumours, depending on the time of AR onset (0-3 weeks after thc start of treatment). ROx of hypoxic tumours resulted in tumoUJ: sensitisation and therefore a dose reduction, of up to 35%, varying with the time of onset. Fig. I shows results for all combinations of AR and ROx onset times for the moderate hypoxia case. Conclusions In hypoxic tumours, accelerated repopulation and reoxy genation affect ccll kill in the same manner as when the effects are modelled individually. however the degree of the effect is altered and therefore the combined result is difficult to predict. providing evidence for the usefulness of computer models. Simulations have quantitatively

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

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Hong, Kyung-Soo [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Park, Jun-Ik [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Dao, Trong Tuan; Oh, Won Keun [BK21 Project Team, College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Kang, Chi-Dug, E-mail: kcdshbw@pusan.ac.kr [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Kim, Sun-Hee, E-mail: ksh7738@pusan.ac.kr [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

2012-03-01

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

International Nuclear Information System (INIS)

Hong, Kyung-Soo; Park, Jun-Ik; Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won; Dao, Trong Tuan; Oh, Won Keun; Kang, Chi-Dug; Kim, Sun-Hee

2012-01-01

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

Effect of hyperthermia and misonidazole on the radiosensitivity of a transplant murine tumor: influence of factors modifying the fraction of hypoxic cells

International Nuclear Information System (INIS)

Wondergem, J.; Haveman, J.; van der Schueren, E.; van den Hoeven, H.; Breur, K.

1982-01-01

Hypoxia has been demonstrated to play an important role in the effect of hyperthermia on tumors. The influence of different factors modifying the oxygenation status of a transplantable murine mammary adenocarcinoma has been studied. The effect of hyperthermia alone on the tumor is not significantly influenced by the change in oxygenation status during the growth of the tumor. Also, the large increase of the acutely hypoxic cell fraction, as a result of anesthesia, does not change the effect of hyperthermia alone. In the combined irradiation-heat treatment there is a clear influence of the chronically hypoxic cell fraction on the response to hyperthermia: an increase in tumor size, resulting in a larger hypoxic cell fraction, leads to an increase in thermal enhancement ratio. However, the increased acutely hypoxic cell fraction, resulting from anesthesia, did not lead to an increase in thermal enhancement ratio; in fact the enhancement ratio apparently decreased. In spite of the fact that hyperthermia was applied immediately after irradiation no potentiation of radiation effects was found. The thermal enhancement of the radiation response was never larger than the enhancement as a result of misonidazole

Regulation of glucose transporter protein-1 and vascular endothelial growth factor by hypoxia inducible factor 1α under hypoxic conditions in Hep-2 human cells.

Science.gov (United States)

Xu, Ou; Li, Xiaoming; Qu, Yongtao; Liu, Shuang; An, Jie; Wang, Maoxin; Sun, Qingjia; Zhang, Wen; Lu, Xiuying; Pi, Lihong; Zhang, Min; Shen, Yupeng

2012-12-01

The present study evaluated the regulation of glucose transporter protein-1 (Glut-1) and vascular endothelial growth factor (VEGF) by hypoxia inducible factor 1α (HIF-1α) under hypoxic conditions in Hep-2 human cells to explore the feasibility of these three genes as tumor markers. Hep-2 cells were cultured under hypoxic and normoxic conditions for 6, 12, 24, 36 and 48 h. The proliferation of Hep-2 cells was evaluated using an MTT assay. The protein and mRNA expression levels of HIF-1α, Glut-1 and VEGF were detected using the S-P immunocytochemical method, western blotting and reverse transcription polymerase chain reaction (RT-PCR). The results revealed that the expression levels of HIF-1α, Glut-1 and VEGF protein in Hep-2 cells were significantly elevated under hypoxic conditions compared with those under normoxic conditions over 36 h. Under hypoxic conditions, mRNA levels of HIF-1α were stable, while mRNA levels of Glut-1 and VEGF changed over time. In conclusion, Glut-1 and VEGF were upregulated by HIF-1α under hypoxic conditions in a time-dependent manner in Hep-2 cells and their co-expression serves as a tumor marker.

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

DEFF Research Database (Denmark)

Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

2011-01-01

Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxi...

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions.

Science.gov (United States)

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

2010-07-16

Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1alpha subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1alpha as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC(50)=5.16microM). The mechanism of this inhibition did not involve suppression of HIF-1alpha protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC(50)=4.75microM). Exposure of Huh7 cells to 10microM kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10microM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent. Copyright 2010 Elsevier Inc. All rights reserved.

The hTERT promoter enhances the antitumor activity of an oncolytic adenovirus under a hypoxic microenvironment.

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Yuuri Hashimoto

Full Text Available Hypoxia is a microenvironmental factor that contributes to the invasion, progression and metastasis of tumor cells. Hypoxic tumor cells often show more resistance to conventional chemoradiotherapy than normoxic tumor cells, suggesting the requirement of novel antitumor therapies to efficiently eliminate the hypoxic tumor cells. We previously generated a tumor-specific replication-competent oncolytic adenovirus (OBP-301: Telomelysin, in which the human telomerase reverse transcriptase (hTERT promoter drives viral E1 expression. Since the promoter activity of the hTERT gene has been shown to be upregulated by hypoxia, we hypothesized that, under hypoxic conditions, the antitumor effect of OBP-301 with the hTERT promoter would be more efficient than that of the wild-type adenovirus 5 (Ad5. In this study, we investigated the antitumor effects of OBP-301 and Ad5 against human cancer cells under a normoxic (20% oxygen or a hypoxic (1% oxygen condition. Hypoxic condition induced nuclear accumulation of the hypoxia-inducible factor-1α and upregulation of hTERT promoter activity in human cancer cells. The cytopathic activity of OBP-301 was significantly higher than that of Ad5 under hypoxic condition. Consistent with their cytopathic activity, the replication of OBP-301 was significantly higher than that of Ad5 under the hypoxic condition. OBP-301-mediated E1A was expressed within hypoxic areas of human xenograft tumors in mice. These results suggest that the cytopathic activity of OBP-301 against hypoxic tumor cells is mediated through hypoxia-mediated activation of the hTERT promoter. Regulation of oncolytic adenoviruses by the hTERT promoter is a promising antitumor strategy, not only for induction of tumor-specific oncolysis, but also for efficient elimination of hypoxic tumor cells.

HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

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Parisa Imanirad

2014-01-01

Full Text Available Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α, a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver, and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.

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

Science.gov (United States)

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

2015-09-01

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

Design of hypoxia-targeting radiopharmaceuticals: selective uptake of copper-64 complexes in hypoxic cells in vitro

International Nuclear Information System (INIS)

Dearling, J.L.J.; Lewis, J.S.; Mullen, G.E.D.; Rae, M.T.; Zweit, J.; Blower, P.J.

1998-01-01

The well-known perfusion tracer CuPTSM, labelled with 62 Cu or 64 Cu, is believed to be trapped in cells non-selectively by a bioreductive mechanism. It is proposed that by modifying the ligand to increase its electron donor strength (for example by adding alkyl functionality or replacing sulphur ligands with oxygen ligands), the copper complexes will become less easily reduced and tracers with selectivity for hypoxic tissues could thus be developed. The aim of this work was to prepare 64 Cu-labelled complexes of two series of ligands, based on the bis(thiosemicarbazone) (13 ligands) and bis(salicylaldimine) (3 ligands) skeletons, and to evaluate the hypoxia dependence of their uptake in cells. The complexes were incubated with Chinese hamster ovary cells under normoxic and hypoxic conditions, and the cells isolated by centrifugation to determine radioactivity uptake at various time points up to 90 min. Several members of both series demonstrated significant (P 60 Cu, 61 Cu, 62 Cu, 64 Cu) and targeted radiotherapy ( 64 Cu, 67 Cu). (orig.)

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

International Nuclear Information System (INIS)

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

2010-01-01

Research highlights: → Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; → Kaempferol causes cytoplasmic mislocalization of HIF-1α by impairing the MAPK pathway. → Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1α subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1α as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC 50 = 5.16 μM). The mechanism of this inhibition did not involve suppression of HIF-1α protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC 50 = 4.75 μM). Exposure of Huh7 cells to 10 μΜ kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 μM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

Energy Technology Data Exchange (ETDEWEB)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas [Laboratory of Biochemistry, School of Medicine, University of Thessaly, BIOPOLIS, 41110 Larissa (Greece); Institute of Biomedical Research and Technology (BIOMED), 51 Papanastasiou str., 41222 Larissa (Greece); Simos, George, E-mail: simos@med.uth.gr [Laboratory of Biochemistry, School of Medicine, University of Thessaly, BIOPOLIS, 41110 Larissa (Greece); Institute of Biomedical Research and Technology (BIOMED), 51 Papanastasiou str., 41222 Larissa (Greece)

2010-07-16

Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

ERRα augments HIF-1 signalling by directly interacting with HIF-1α in normoxic and hypoxic prostate cancer cells.

Science.gov (United States)

Zou, Chang; Yu, Shan; Xu, Zhenyu; Wu, Dinglan; Ng, Chi-Fai; Yao, Xiaoqiang; Yew, David T; Vanacker, Jean-Marc; Chan, Franky L

2014-05-01

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up-regulation of hypoxia-inducible factor 1 (HIF-1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen-related receptor-α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα-over-expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF-1α protein expression and HIF-1 signalling. These effects could also be observed in ERRα-over-expressing cells grown under normoxia, suggesting that ERRα could function to pre-adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF-1α via its AF-2 domain. A ubiquitination assay showed that this ERRα-HIF-1α interaction could inhibit ubiquitination of HIF-1α and thus reduce its degradation. Such ERRα-HIF-1α interaction could be attenuated by XCT790, an ERRα-specific inverse agonist, resulting in reduced HIF-1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF-1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions

Science.gov (United States)

Felfoul, Ouajdi; Mohammadi, Mahmood; Taherkhani, Samira; de Lanauze, Dominic; Zhong Xu, Yong; Loghin, Dumitru; Essa, Sherief; Jancik, Sylwia; Houle, Daniel; Lafleur, Michel; Gaboury, Louis; Tabrizian, Maryam; Kaou, Neila; Atkin, Michael; Vuong, Té; Batist, Gerald; Beauchemin, Nicole; Radzioch, Danuta; Martel, Sylvain

2016-11-01

Oxygen-depleted hypoxic regions in the tumour are generally resistant to therapies. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour of magnetotactic bacteria, Magnetococcus marinus strain MC-1 (ref. 4), can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals, tend to swim along local magnetic field lines and towards low oxygen concentrations based on a two-state aerotactic sensing system. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in severe combined immunodeficient beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1.

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Xue, Mei; Chen, Wei; Xiang, An; Wang, Ruiqi; Chen, He; Pan, Jingjing; Pang, Huan; An, Hongli; Wang, Xiang; Hou, Huilian; Li, Xu

2017-08-25

To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number of non-coding RNA-containing exosomes that facilitate tumor development and metastasis. However, the precise mechanisms of tumor cell-derived exosomes during hypoxia are unknown. Here, we aim to clarify whether hypoxia affects tumor growth and progression by transferring long non-coding RNA-urothelial cancer-associated 1 (lncRNA-UCA1) enriched exosomes secreted from bladder cancer cells. We used bladder cancer 5637 cells with high expression of lncRNA-UCA1 as exosome-generating cells and bladder cancer UMUC2 cells with low expression of lncRNA-UCA1 as recipient cells. Exosomes derived from 5637 cells cultured under normoxic or hypoxic conditions were isolated and identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting analysis. These exosomes were co-cultured with UMUC2 cells to evaluate cell proliferation, migration and invasion. We further investigated the roles of exosomal lncRNA-UCA1 derived from hypoxic 5637 cells by xenograft models. The availability of lncRNA-UCA1 in serum-derived exosomes as a biomarker for bladder cancer was also assessed. We found that hypoxic exosomes derived from 5637 cells promoted cell proliferation, migration and invasion, and hypoxic exosomal RNAs could be internalized by three bladder cancer cell lines. Importantly, lncRNA-UCA1 was secreted in hypoxic 5637 cell-derived exosomes. Compared with normoxic exosomes, hypoxic exosomes derived from 5637 cells showed the higher expression levels of lncRNA-UCA1. Moreover, Hypoxic exosomal lncRNA-UCA1 could promote tumor growth and progression though epithelial-mesenchymal transition, in vitro and in vivo. In addition, the expression levels of lncRNA-UCA1 in the human serum-derived exosomes of bladder cancer patients were higher than that in the healthy controls. Together, our results demonstrate that hypoxic bladder cancer cells remodel tumor

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

Directory of Open Access Journals (Sweden)

Yufen Xie

2014-11-01

Full Text Available Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC, elevated O2 and mitochondrial function are necessary to placental lineages at the maternal–placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. In an implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor 4 (FGF4 enabled the highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2 initiated the most TSC differentiation after 24 h despite exposure to FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential and maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos, and high pyruvate kinase M2 (glycolysis despite FGF4 removal. Inhibiting OxPhos inhibited optimum differentiation at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2 > 0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation.

Effects of the differentiating agents sodium butyrate and N-methylformamide on the oxygen enhancement ratio of human colon tumor cells

International Nuclear Information System (INIS)

Hallows, K.R.; Bliven, S.F.; Leith, J.T.

1988-01-01

We have previously shown that chronic adaptation of human tumor cells to the differentiation-inducing agents N-methylformamide (NMF) and sodium butyrate (NAB) increases the sensitivity of oxic cells to graded single doses of X rays. These studies were carried out to define the sensitivity of hypoxic cells after adaptation. Clone A colon tumor cells were grown for three passages in medium containing 170 mM NMF or 2 mM NAB and irradiated in suspension culture, after gassing with either oxygen (60 min) or ultrapure nitrogen (90 min), and complete survival curves were generated. Using the linear-quadratic equation to describe the data, it was found that NMF and NAB produced increased X-ray killing of hypoxic cells. At the 10% level of survival, the dose-modifying factors were about 1.20 and 1.25 for NMF- and NAB-adapted hypoxic cells, respectively, as compared to hypoxic control cells. However, since both oxic and hypoxic cells exhibited increased sensitivity after NMF and NAB adaptation, there was no major change in the oxygen enhancement ratio

Hypoxic cytotoxicity of chlorpromazine and the modification of radiation response in E. coli B/r

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Shenoy, M.A.; Singh, B.B.

1978-01-01

Chlorpromazine (0.1 mM) was cytotoxic to E. coli B/r cells under hypoxic but not euoxic conditions. Under nitrogen bubbling, there was no further enhancement in cellular lethality beyond 45 min contact time. The presence of the free drug seemed necessary for the cytocidal action to be demonstrated. Hypoxic cytotoxicity increased steadily with temperature between 30 and 37 0 C. Treatment of cells with N-ethyl maleimide (0.5 mM) completely abolished the subsequent hypoxic cytotoxicity of chlorpromazine (0.1 mM). Hypoxic gamma irradiation of cells pretreated for 45 min with chlorpromazine under nitrogen bubbling gave a DMF for survival of almost twice that produced by oxygen. Irradiation under aerobic conditions of cells subjected to the same pretreatment produced only the normal oxygen effect. The results indicate that the differential cytotoxicity of chlorpromazine is due to its effect on the changes induced in the membrane-associated biochemical state of the cells under euoxic and hypoxic conditions. (U.K.)

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans.

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Sun, C-L; Kim, E; Crowder, C M

2014-04-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

Interaction of nitroimidazole sensitizers and oxygen in the radiosensitization of mammalian cells at ultrahigh dose rates

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Michaels, H.B.; Ling, C.C.; Epp, E.R.; Peterson, E.C.

1981-01-01

When CHO cells, equilibrated with 0.44% oxygen, are irradiated with single 3-nsec pulses of electrons from a 600-kV-field emission source, a breaking survival curve is observed. The breaking behavior, believed to be the result of radiolytic oxygen depletion, can be prevented by the presence of a relatively low concentration of the hypoxic cell sensitizer misonidazole; similar results are obtained with metronidazole and Ro-05-9963. The resulting survival curves exhibit a sensitized response similar to that obtained with conventional dose rate radiation for CHO cells under this oxygen concentration. This degree of sensitization is greater than that observed for CHO cells irradiated at ultrahigh dose rates under the same concentration of sensitizer in nitrogen. The data suggest that the nitroimidazole compounds interfere with the radiation chemical oxygen depletion process and that the radiosensitization observed in the nonbreaking survival curve is the consequence of sensitization by both the nitroimidazole and, primarily, the oxygen rather than a direct subsitution for oxygen by the sensitizer. This conclusion is also supported by data obtained in double-pulse experiments. The results are discussed with regard to the mechanisms of the oxygen depletion process and radiosensitization

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Science.gov (United States)

Takizawa, Naoki; Okubo, Naoto; Kamo, Masaharu; Chosa, Naoyuki; Mikami, Toshinari; Suzuki, Keita; Yokota, Seiji; Ibi, Miho; Ohtsuka, Masato; Taira, Masayuki; Yaegashi, Takashi; Ishisaki, Akira; Kyakumoto, Seiko

2017-09-15

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Decursin reduce radio-resistance of hypoxic regions under the proton beam therapy by induced HIF-1α degradation

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Jung, Myung Hwan; Kim, Kye Ryung

2013-01-01

Protons induce cancer-cell apoptosis in vitro and block blood vessel formation in vivo through the generation of reactive oxygen species (ROS). The fact that proton severely inhibits blood vessel development in zebrafish embryos suggests a higher sensitivity of vascular endothelial cells to proton beam. Decursin, a coumarin compound, was originally isolated from Angelica gigas Nakai (Dang Gui). A. gigas root has been traditionally used in Korean folk medicine for the treatment of anemia and other common diseases. In previous reports, decursin was reported to exhibit anti-tumor activity against various cancer cells and to inhibit the activities of the androgen and androgen-receptor (AR) signaling pathway in prostate cancer, induction of cell cycle arrest and apoptosis in various cancer cells, such as prostate, breast, bladder, and colon cancer cells. Decursin also inhibits VEGF-induced angiogenesis through the suppression of the VEGFR-2-signaling pathway. However, the mechanism of decursin mediates change of HIF-1α activities is not clear. In this research, we identified regulations of the HIF-1α and the anti-angiogenesis effects of decursin in proton-beam-irradiated human lung cancer, prostate cancer and Hepatic cancer cells. We investigated the underlying mechanisms of positive effects of protonbeam-induced anti-angiogenesis. Our data indicate that the groups co-treated with decursin and a proton-beam had significant reduced HIF-1α activity compared with the groups treated with only a proton beam under the hypoxic condition caused by DFX(desferrioxamine). Decursin was found to induced HIF-1α degradation. Therefore, we suggest that decursin may be a potential candidate for use as a sensitizer for proton-beaminduced cell apoptosis. Here we have shown that decursin successfully reduced HIF-1α stability under hypoxic condition by induced desferrioxamine. We showed novel candidates for anti-angiogenic compound, decursin, leading to complete inhibition of radio

Decursin reduce radio-resistance of hypoxic regions under the proton beam therapy by induced HIF-1α degradation

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Jung, Myung Hwan; Kim, Kye Ryung [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2013-10-15

Protons induce cancer-cell apoptosis in vitro and block blood vessel formation in vivo through the generation of reactive oxygen species (ROS). The fact that proton severely inhibits blood vessel development in zebrafish embryos suggests a higher sensitivity of vascular endothelial cells to proton beam. Decursin, a coumarin compound, was originally isolated from Angelica gigas Nakai (Dang Gui). A. gigas root has been traditionally used in Korean folk medicine for the treatment of anemia and other common diseases. In previous reports, decursin was reported to exhibit anti-tumor activity against various cancer cells and to inhibit the activities of the androgen and androgen-receptor (AR) signaling pathway in prostate cancer, induction of cell cycle arrest and apoptosis in various cancer cells, such as prostate, breast, bladder, and colon cancer cells. Decursin also inhibits VEGF-induced angiogenesis through the suppression of the VEGFR-2-signaling pathway. However, the mechanism of decursin mediates change of HIF-1α activities is not clear. In this research, we identified regulations of the HIF-1α and the anti-angiogenesis effects of decursin in proton-beam-irradiated human lung cancer, prostate cancer and Hepatic cancer cells. We investigated the underlying mechanisms of positive effects of protonbeam-induced anti-angiogenesis. Our data indicate that the groups co-treated with decursin and a proton-beam had significant reduced HIF-1α activity compared with the groups treated with only a proton beam under the hypoxic condition caused by DFX(desferrioxamine). Decursin was found to induced HIF-1α degradation. Therefore, we suggest that decursin may be a potential candidate for use as a sensitizer for proton-beaminduced cell apoptosis. Here we have shown that decursin successfully reduced HIF-1α stability under hypoxic condition by induced desferrioxamine. We showed novel candidates for anti-angiogenic compound, decursin, leading to complete inhibition of radio

SUMO Signaling by Hypoxic Inactivation of SUMO-Specific Isopeptidases

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Kathrin Kunz

2016-09-01

Full Text Available Post-translational modification of proteins with ubiquitin-like SUMO modifiers is a tightly regulated and highly dynamic process. The SENP family of SUMO-specific isopeptidases comprises six cysteine proteases. They are instrumental in counterbalancing SUMO conjugation, but their regulation is not well understood. We demonstrate that in hypoxic cell extracts, the catalytic activity of SENP family members, in particular SENP1 and SENP3, is inhibited in a rapid and fully reversible process. Comparative mass spectrometry from normoxic and hypoxic cells defines a subset of hypoxia-induced SUMO1 targets, including SUMO ligases RanBP2 and PIAS2, glucose transporter 1, and transcriptional regulators. Among the most strongly induced targets, we identified the transcriptional co-repressor BHLHE40, which controls hypoxic gene expression programs. We provide evidence that SUMOylation of BHLHE40 is reversed by SENP1 and contributes to transcriptional repression of the metabolic master regulator gene PGC-1α. We propose a pathway that connects oxygen-controlled SENP activity to hypoxic reprogramming of metabolism.

Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

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Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

2011-04-15

Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

International Nuclear Information System (INIS)

Staab, Adrian; Fleischer, Markus; Wuerzburg Univ.; Loeffler, Juergen; Einsele, Herrmann; Said, Harun M.; Katzer, Astrid; Flentje, Michael; Plathow, Christian; Vordermark, Dirk; Halle-Wittenberg Univ.

2011-01-01

Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O 2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O 2 as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

Fetal Stress and Programming of Hypoxic/Ischemic-Sensitive Phenotype in the Neonatal Brain: Mechanisms and Possible Interventions

Science.gov (United States)

Li, Yong; Gonzalez, Pablo; Zhang, Lubo

2012-01-01

Growing evidence of epidemiological, clinical and experimental studies has clearly shown a close link between adverse in utero environment and the increased risk of neurological, psychological and psychiatric disorders in later life. Fetal stresses, such as hypoxia, malnutrition, and fetal exposure to nicotine, alcohol, cocaine and glucocorticoids may directly or indirectly act at cellular and molecular levels to alter the brain development and result in programming of heightened brain vulnerability to hypoxic-ischemic encephalopathy and the development of neurological diseases in the postnatal life. The underlying mechanisms are not well understood. However, glucocorticoids may play a crucial role in epigenetic programming of neurological disorders of fetal origins. This review summarizes the recent studies about the effects of fetal stress on the abnormal brain development, focusing on the cellular, molecular and epigenetic mechanisms and highlighting the central effects of glucocorticoids on programming of hypoxicischemic-sensitive phenotype in the neonatal brain, which may enhance the understanding of brain pathophysiology resulting from fetal stress and help explore potential targets of timely diagnosis, prevention and intervention in neonatal hypoxic-ischemic encephalopathy and other for brain disorders. PMID:22627492

Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells.

Science.gov (United States)

Filipp, Fabian V; Scott, David A; Ronai, Ze'ev A; Osterman, Andrei L; Smith, Jeffrey W

2012-05-01

The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

Science.gov (United States)

Ghourichaee, Sasan Sharee; Powell, Elizabeth M; Leach, Jennie B

2017-05-01

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O 2 ) versus hypoxic (3% O 2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition.

Science.gov (United States)

Minchenko, D O; Riabovol, O O; Ratushna, O O; Minchenko, O H

2017-01-01

The aim of the present study was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoded estrogen related proteins (NRIP1/RIP140, TRIM16/EBBP, ESRRA/NR3B1, FAM162A/E2IG5, PGRMC2/PMBP, and SLC39A6/LIV-1) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of glioma cells proliferation. The expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by a quantitative polymerase chain reaction. Inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 signaling enzyme function up-regulates the expression of EBBP, E2IG5, PGRMC2, and SLC39A6 genes is in U87 glioma cells in comparison with the control glioma cells, with more significant changes for E2IG5 and PGRMC2 genes. At the same time, the expression of NRIP1 and ESRRA genes is strongly down-regulated in glioma cells upon inhibition of IRE1. We also showed that hypoxia increases the expression of E2IG5, PGRMC2, and EBBP genes and decreases NRIP1 and ESRRA genes expression in control glioma cells. Furthermore, the inhibition of IRE1 in U87 glioma cells decreases the eff ect of hypoxia on the expression of E2IG5 and PGRMC2 genes, eliminates hypoxic regulation of NRIP1 gene, and enhances the sensitivity of ESRRA gene to hypoxic condition. Furthermore, the expression of SLC39A6 gene is resistant to hypoxia in both the glioma cells with and without IRE1 signaling enzyme function. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in gene specific manner and these changes

[Sensitivity and specificity of the cerebral blood flow reactions to acupuncture in the newborn infants presenting with hypoxic ischemic encephalopathy].

Science.gov (United States)

Filonenko, A V; Vasilenko, A M; Khan, M A

2015-01-01

To evaluate the effects of acupuncture integrated into the standard therapy, the condition of cerebral blood flow, and other syndromes associated with cerebral ischemia in the newborn infants. MATERIAL AND METHODS. A total of 131 pairs of puerperae and newborns with hypoxic ischemic encephalopathy were divided into four treatment groups. 34 children of the first group were given standard therapy (control), in the second group comprised of 33 mothers and children the standard treatment was supplemented by acupuncture, the third group included only 32 mothers given the acupuncture treatment alone, and the fourth group contained only 32 newborn infants treated by acupuncture. Each course of acupuncture treatment consisted of five sessions. Sensitivity and specificity of cerebral blood flow reactions were determined based on the results of the ROC-analysis and the area under the curve before and after the treatment. The treatment with the use of acupuncture greatly improved the cerebrospinal hemodynamics (p newborn babies. The high level of sensitivity (84.4-94.8%) associated with good specificity makes it possible to distinguish between the true positive and true negative cases. Acupuncture integrated into the treatment of "mother-baby" pairs presenting with hypoxic ischemic encephalopathy can be used to improve the initially low level of cerebral blood flow in neonates presenting with this condition.

Hypoxic Response of Tumor Tissues in a Microfluidic Environment

Science.gov (United States)

Morshed, Adnan; Dutta, Prashanta

2017-11-01

Inside a tumor tissue, cells growing further away from the blood vessel often suffer from low oxygen levels known as hypoxia. Cancer cells have shown prolonged survival in hostile hypoxic conditions by sharply changing the cellular metabolism. In this work, different stages of growth of the tumor tissue and the oxygen transport across the tissue are investigated. The tissue was modeled as a contiguous block of cells inside a microfluidic environment with nutrient transport through advection and diffusion. While oxygen uptake inside the tissue is through diffusion, ascorbate transport from the extracellular medium is addressed by a concentration dependent uptake model. By varying the experimentally observed oxygen consumption rate, different types of cancer cells and their normoxic and hypoxic stages were studied. Even when the oxygen supply in the channel is maintained at normoxic levels, our results show the onset of hypoxia within minutes inside the cellblock. Interestingly, modeled cell blocks with and without a structured basal layer showed less than 5% variation in hypoxic response in chronic hypoxia. Results also indicate that the balance of cell survival and growth are affected by the flow rate of nutrients and the oxygen consumption rate. This work was supported in part by the National Science Foundation under Grant No. DMS 1317671.

[The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

Science.gov (United States)

Poleshko, A G; Lobanok, E S; Mezhevikina, L M; Fesenko, E E; Volotkovskiĭ, I D

2014-01-01

It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

MicroRNA response to hypoxic stress in soft tissue sarcoma cells: microRNA mediated regulation of HIF3α

International Nuclear Information System (INIS)

Gits, Caroline MM; Wiemer, Erik AC; Kuijk, Patricia F van; Rijck, Jonneke CWM de; Muskens, Nikky; Jonkers, Moniek BE; IJcken, Wilfred F van; Mathijssen, Ron HJ; Verweij, Jaap; Sleijfer, Stefan

2014-01-01

Hypoxia is often encountered in solid tumors and known to contribute to aggressive tumor behavior, radiation- and chemotherapy resistance resulting in a poor prognosis for the cancer patient. MicroRNAs (miRNAs) play a role in the regulation of the tumor cell response to hypoxia, however, not much is known about the involvement of miRNAs in hypoxic signalling pathways in soft tissue sarcomas (STS). A panel of twelve STS cell lines was exposed to atmospheric oxygen concentrations (normoxia) or 1% oxygen (hypoxia) for up to 48 h. Hypoxic conditions were verified and miRNA expression profiles were assessed by LNA™ oligonucleotide microarrays and RT-PCR after 24 h. The expression of target genes regulated by hypoxia responsive miRNAs is examined by end-point PCR and validated by luciferase reporter constructs. Exposure of STS cell lines to hypoxic conditions gave rise to upregulation of Hypoxia Inducible Factor (HIF) 1α protein levels and increased mRNA expression of HIF1 target genes CA9 and VEGFA. Deregulation of miRNA expression after 24 h of hypoxia was observed. The most differentially expressed miRNAs (p < 0.001) in response to hypoxia were miR-185-3p, miR-485-5p, miR-216a-5p (upregulated) and miR-625-5p (downregulated). The well-known hypoxia responsive miR-210-3p could not be reliably detected by the microarray platform most likely for technical reasons, however, its upregulation upon hypoxic stress was apparent by qPCR. Target prediction algorithms identified 11 potential binding sites for miR-485-5p and a single putative miR-210-3p binding site in the 3’UTR of HIF3α, the least studied member of the HIF family. We showed that HIF3α transcripts, expressing a 3’UTR containing the miR-485-5p and miR-210-3p target sites, are expressed in all sarcoma cell lines and upregulated upon hypoxia. Additionally, luciferase reporter constructs containing the 3’UTR of HIF3α were used to demonstrate regulation of HIF3α by miR-210-3p and miR-485-5p. Here we provide

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

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

2003-01-01

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

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

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Santhosh, K T; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, A J; Dakshinamurti, S

2011-07-01

Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

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

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Xia, Tian; Cheng, Hao; Zhu, Yu

2014-01-01

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

How far is cancer cured by radiation sensitization?

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Ando, Koichi; Sasaki, Takehito; Ikeda, Hiroshi

1990-01-01

Some types of cancer are not cured by radiation alone in view of histology, location, and size. In facing so-called radioresistant cancer, antineoplastic agents, hypoxic cell sensitizers, biological response modifiers, or hyperthermia are used in combination with radiation, with the aim of cancer cure. First of all, this chapter discusses the subject of 'what is tumor cure by radiation therapy'. Current conditions of the aforementioned combined modalities and the future perspectives are presented. The following subjects are covered: (1) tumor control - significance of the number of stem cells; (2) biological evaluation of chemo-radiotherapy with cisplatin; (3) clinical results and experience with combination of radiotherapy and radiosensitizers; (4) radiosensitization with hypoxic cell radiosensitizers - present status (5) hypoxic cell radiosensitizers - present status and problems from the viewpoint of clinical radiotherapy; (6) thermal radiosensitization in vitro and its implications for radiotherapy; (7) clinical assessment of thermoradiotherapy for breast cancer and cancer of the urinary bladder; (8) interactions of radiation and biological response modifiers in the treatment of malignant tumor; (9) improvement in the effects of radiation therapy with biological response modifiers. (N.K.)

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.

Effects of hypoxic preconditioning on the changes of expression of neuroglobin mRNA and labeled positive cells following cerebral ischemia in gerbils

Institute of Scientific and Technical Information of China (English)

Yong Zhang; Yanqun Chang; Zhenfang Liu; Qingxi Fu; Xiao Zhang

2006-01-01

BACKGROUND: Neuroglobin (NGB), as newly discovered the third member of the globin family binding oxygen, mainly exists in brain of human beings and vertebrates, and it is closely correlated with the oxygen supply in brain.OBJECTIVE : To observe the changes of the expression of neuroglobin and number of positive cells labeled immunohistochemically following cerebral ischemia in gerbils after hypoxic preconditioning. DESIGN: A complete randomized grouping design and controlled experiment. SETTING: Department of Neurology, Department of Anesthesia, Shandong Provincial Hospital, Shandong University.MATERIALS: Sixty-six adult male Mongolian gerbils of clean degree, about 50-65 g, at an average of 57.5 g were provided by the Experimental Animal Center of Capital Medical University [certificate number of animal quality:SCXK(Beijing)2000-0012]. TRNzil (Tianwei Shidai Company, Beijing), polymerase chain reaction (PCR) primers (synthetized by Invitrogen Company, Shanghai); reverse transcription-PCR (RT-PCR) one-step kit (Toyobo Company); PCR instrument (GeneAmp PCR System 240); mice brain NGB monoclonal antibody (Academy of Military Medical Sciences); DAB (Zhongshan Company, Beijing). METHEDS: The study was completed from December 2004 to June 2005 in Shandong Provincial Hospital. ① The 66 gerbils were randomly divided into sham-operated group (n =6), cerebral ischemia group (n =30) and hypoxic preconditioning group (n =30). The gerbils in the hypoxic preconditioning group were put in the environment which contained O2 (0.08 in volume fraction) and N2 (0.92 in volume fraction) at temperature of 25 ℃ for 2 hours. After 5 hours, the gerbils in the hypoxic preconditioning group and cerebral ischemia group were anesthetized, then bilateral common carotid arteries were ligated. In the sham-operated group, bilateral common carotid arteries were only isolated without ligation and hypoxic preconditioning. ②2 At 1, 5, 10, 30 and 60 minutes after cerebral ischemia, the

Understanding Hypoxic Drive and the Release of Hypoxic Vasoconstriction.

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Inkrott, Jon C

2016-01-01

Understanding the hypoxic drive and release of hypoxic vasoconstriction in the chronic obstructive pulmonary disease population can be somewhat confusing and misunderstood. Furthermore, the hypoxic drive theory is one in which there really is no scientific evidence to support and yet continues to prosper in every aspect of care in regard to the chronic lung patient, from prehospital all the way to intensive care unit and home care therapy. This subject review will hopefully enhance some understanding of what exactly goes on with these patients and the importance of providing oxygen when it is desperately needed. Copyright © 2016 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.

mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells.

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Kuo, Chung-Wen; Tsai, Meng-Han; Lin, Tsu-Kung; Tiao, Mao-Meng; Wang, Pei-Wen; Chuang, Jiin-Haur; Chen, Shang-Der; Liou, Chia-Wei

2017-06-07

Mitochondria consume O₂ to produce ATP and are critical for adaption of hypoxia, but the role of mitochondria in HIF-1α pathway is as yet unclear. In this study, mitochondrial DNA (mtDNA) enriched (SK-N-AS) and depleted (ρ⁰) cells of neuroblastoma were cultured in a hypoxic chamber to simulate a hypoxic condition and then the major components involved in mitochondrial related pathways, hypoxia-inducible factor 1α (HIF-1α) and reactive oxygen species (ROS) were measured. The results showed that hypoxia-stimulated exposure elevated expression of HIF-1α, which was additionally influenced by level of generated ROS within the cytosol. Moreover, elevation of HIF-1α also resulted in increases of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase kinase 1 (PDK1) in both hypoxic cells. The expression of mitochondrial biogenesis related proteins and metabolic components were noted to increase significantly in hypoxic SK-N-AS cells, indicating that mtDNA was involved in mitochondrial retrograde signaling and metabolic pathways. An analysis of dynamic proteins found elevated levels of HIF-1α causing an increased expression of dynamin-related protein 1 (DRP1) during hypoxia; further, the existence of mtDNA also resulted in higher expression of DRP1 during hypoxia. By using siRNA of HIF-1α or DRP1, expression of DRP1 decreased after suppression of HIF-1α; moreover, the expression of HIF-1α was also affected by the suppression of DRP1. In this study, we demonstrated that mtDNA is a mediator of HIF-1α in eliciting metabolic reprogramming, and mitochondrial biogenesis. Identification of a mutual relationship between HIF-1α and DRP1 may be a critical tool in the future development of clinical applications.

The role of heat shock protein (HSP as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture

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Sri Wigati Mardi Mulyani

2014-03-01

Full Text Available Background: The concept of stem cell therapy is one of the new hope as a medical therapy on salivary gland defect. However, the lack of viability of the transplanted stem cells survival rate led to the decrease of effectiveness of stem cell therapy. The underlying assumption in the decrease of viability and function of stem cells is an increase of apoptosis incidence. It suggests that the microenvironment in the area of damaged tissues is not conducive to support stem cell viability. One of the microenvironment is the hypoxia condition. Several scientific journals revealed that the administration of hypoxic cell culture can result in stress cells but on the other hand the stress condition of the cells also stimulates heat shock protein 27 (HSP 27 as antiapoptosis through inhibition of caspase 9. Purpose: The purpose of this study was to examine the role of heat shock protein 27 as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture. Methods: Stem cell culture was performed in hypoxic conditions (O2 1% and measured the resistance to apoptosis through HSP 27 and caspase 9 expression of bone marrow mesenchymal stem cells by using immunoflorecence and real time PCR. Results: The result of study showed that preconditioning hypoxia could inhibit apoptosis through increasing HSP 27 and decreasing level of caspase 9. Conclusion: The study suggested that hypoxic precondition could reduce apoptosis by increasing amount of heat shock protein 27 and decreasing caspase 9.Latar belakang: Konsep terapi stem cell merupakan salah satu harapan baru sebagai terapi medis kelainan kelenjar ludah. Namun, rendahnya viabilitas stem cell yang ditransplantasikan menyebabkan penurunan efektivitas terapi. Asumsi yang mendasari rendahnya viabilitas dan fungsi stem cell adalah tingginya kejadian apoptosis. Hal ini menunjukkan bahwa lingkungan mikro di daerah jaringan yang rusak tidak kondusif untuk mendukung viabilitas stem cell. Salah satu lingkungan

Gold namoprtices enhance anti-tumor effect of radiotherapy to hypoxic tumor

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Kim, Mi Sun; Lee, Eun Jung; Kim, Jae Won; Keum, Ki Chang; Koom, Woong Sub [Dept. of Radiation Oncology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Chung, Ui Seok; Koh, Won Gun [Dept. of Chemical and Biomolecular Engineering, Yonsei University, Seoul (Korea, Republic of)

2016-09-15

Hypoxia can impair the therapeutic efficacy of radiotherapy (RT). Therefore, a new strategy is necessary for enhancing the response to RT. In this study, we investigated whether the combination of nanoparticles and RT is effective in eliminating the radioresistance of hypoxic tumors. Gold nanoparticles (GNPs) consisting of a silica core with a gold shell were used. CT26 colon cancer mouse model was developed to study whether the combination of RT and GNPs reduced hypoxia-induced radioresistance. Hypoxia inducible factor-1α (HIF-1α) was used as a hypoxia marker. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were conducted to evaluate cell death. Hypoxic tumor cells had an impaired response to RT. GNPs combined with RT enhanced anti-tumor effect in hypoxic tumor compared with RT alone. The combination of GNPs and RT decreased tumor cell viability compare to RT alone in vitro. Under hypoxia, tumors treated with GNPs + RT showed a higher response than that shown by tumors treated with RT alone. When a reactive oxygen species (ROS) scavenger was added, the enhanced antitumor effect of GNPs + RT was diminished. In the present study, hypoxic tumors treated with GNPs + RT showed favorable responses, which might be attributable to the ROS production induced by GNPs + RT. Taken together, GNPs combined with RT seems to be potential modality for enhancing the response to RT in hypoxic tumors.

Gold namoprtices enhance anti-tumor effect of radiotherapy to hypoxic tumor

International Nuclear Information System (INIS)

Kim, Mi Sun; Lee, Eun Jung; Kim, Jae Won; Keum, Ki Chang; Koom, Woong Sub; Chung, Ui Seok; Koh, Won Gun

2016-01-01

Hypoxia can impair the therapeutic efficacy of radiotherapy (RT). Therefore, a new strategy is necessary for enhancing the response to RT. In this study, we investigated whether the combination of nanoparticles and RT is effective in eliminating the radioresistance of hypoxic tumors. Gold nanoparticles (GNPs) consisting of a silica core with a gold shell were used. CT26 colon cancer mouse model was developed to study whether the combination of RT and GNPs reduced hypoxia-induced radioresistance. Hypoxia inducible factor-1α (HIF-1α) was used as a hypoxia marker. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were conducted to evaluate cell death. Hypoxic tumor cells had an impaired response to RT. GNPs combined with RT enhanced anti-tumor effect in hypoxic tumor compared with RT alone. The combination of GNPs and RT decreased tumor cell viability compare to RT alone in vitro. Under hypoxia, tumors treated with GNPs + RT showed a higher response than that shown by tumors treated with RT alone. When a reactive oxygen species (ROS) scavenger was added, the enhanced antitumor effect of GNPs + RT was diminished. In the present study, hypoxic tumors treated with GNPs + RT showed favorable responses, which might be attributable to the ROS production induced by GNPs + RT. Taken together, GNPs combined with RT seems to be potential modality for enhancing the response to RT in hypoxic tumors

Low-dose radiation suppresses Pokemon expression under hypoxic conditions.

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Kim, Seung-Whan; Yu, Kweon; Shin, Kee-Sun; Kwon, Kisang; Hwang, Tae-Sik; Kwon, O-Yu

2014-01-01

Our previous data demonstrated that CoCl2-induced hypoxia controls endoplasmic reticulum (ER) stress-associated and other intracellular factors. One of them, the transcription factor Pokemon, was differentially regulated by low-dose radiation (LDR). There are limited data regarding how this transcription factor is involved in expression of the unfolded protein response (UPR) under hypoxic conditions. The purpose of this study was to obtain clues on how Pokemon is involved in the UPR. Pokemon was selected as a differentially expressed gene under hypoxic conditions; however, its regulation was clearly repressed by LDR. It was also demonstrated that both expression of ER chaperones and ER stress sensors were affected by hypoxic conditions, and the same results were obtained when cells in which Pokemon was up- or down-regulated were used. The current state of UPR and LDR research associated with the Pokemon pathway offers an important opportunity to understand the oncogenesis, senescence, and differentiation of cells, as well as to facilitate introduction of new therapeutic radiopharmaceuticals.

Dinitrobenzamide mustard prodrugs - hypoxic cytotoxins and dual substrates for E.coli nitroreductase

International Nuclear Information System (INIS)

Patterson, A.V.; Hogg, A.; Pullen, S.; Degenkolbe, A.; Li, D.; Chappell, A.; Ying, S.; Atwell, G.J.; Denny, W.A.; Anderson, R.F.; Wilson, W.R.

2003-01-01

Conditional replicating adenoviral vectors (CRAds) have received considerable attention as therapeutic tools in combination with radiotherapy. Viral distribution and micro-regional geometry are likely to be important issues in the treatment of human solid tumours with gene therapy, particularly following intravenous virus administration. The use of CRAds that are 'armed' with enzyme/prodrug systems may overcome some of the perceived limitations; CRAds can redistribute and self-amplify in a cytolytic fashion whilst prodrug metabolites may elicit a local bystander effect. Either or both of these cytotoxic properties could have favourable interactions with radiotherapy (IR). Nevertheless, they may be insufficient to avoid pockets of vector-naive tumour cells beyond the diffusion limits of cytotoxic prodrug metabolites, such as when perivascular seeding occurs. Under such circumstances hypoxic tumour cells may represent the least accessible compartment for vector transfection; the same tumour subpopulation that is likely to be radioresistant. E.coli nitroreductase (NTR) can bioactivate dinitrobenzamide mustards (DNBMs) and is a promising enzyme/prodrug system for 'arming' CRAds. Notably DNMBs can also be activated by endogenous human reductases under low oxygen conditions providing an opportunity to identify dual hypoxic cytotoxins/NTR substrates that may circumvent some of the geometry issues and provide complementarity with IR. To identify a prodrug for NTR that is also active as a hypoxic cytotoxin in vivo. From a set of 164 DNB prodrugs, 19 with favourable activity in vitro against a panel of four NTR-expressing cancer cells were selected and screened for activity as hypoxic cytotoxins in vitro. Measured E17 values ranged from -444 to -366 mV. Seven DNBMs possessed acceptable hypoxic selectivity against the human NSCLC cell line A549WT or clones engineered to overexpress either a human single-electron reductase, cytochrome P450 reductase (A549P450R), or oxic

An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors.

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Szablowski, Jerzy O; Raskatov, Jevgenij A; Dervan, Peter B

2016-04-01

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1: has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1: in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1: distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1: show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response. Mol Cancer Ther; 15(4); 608-17. ©2015 AACR. ©2015 American Association for Cancer Research.

Introduction to altitude/hypoxic training symposium.

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Wilber, Randall L

2007-09-01

Altitude/hypoxic training has traditionally been an intriguing and controversial area of research and sport performance. This controversial aspect was evident recently in the form of scholarly debates in highly regarded professional journals, as well as the World Anti-Doping Agency's (WADA) consideration of placing "artificially-induced hypoxic conditions" on the 2007 Prohibited List of Substances/Methods. In light of the ongoing controversy surrounding altitude/hypoxic training, this symposium was organized with the following objectives in mind: 1) to examine the primary physiological responses and underlying mechanisms associated with altitude/hypoxic training, including the influence of genetic predisposition; 2) to present evidence supporting the effect of altitude/hypoxic acclimatization on both hematological and nonhematological markers, including erythrocyte volume, skeletal muscle-buffering capacity, hypoxic ventilatory response, and physiological efficiency/economy; 3) to evaluate the efficacy of several contemporary simulated altitude modalities and training strategies, including hypoxic tents, nitrogen apartments, and intermittent hypoxic exposure (IHE) or training, and to address the legal and ethical issues associated with the use of simulated altitude; and 4) to describe different altitude/hypoxic training strategies used by elite-level athletes, including Olympians and military special forces. In addressing these objectives, papers will be presented on the topics of: 1) effect of hypoxic "dose" on physiological responses and sea-level performance (Drs. Benjamin Levine and James Stray-Gundersen), 2) nonhematological mechanisms of improved performance after hypoxic exposure (Dr. Christopher Gore), 3) application of altitude/hypoxic training by elite athletes (Dr. Randall Wilber), and 4) military applications of hypoxic training (Dr. Stephen Muza).

Glyoxylic compounds as radiosensitizers of hypoxic cells

International Nuclear Information System (INIS)

Cornago, M.P.; Lopez Zumel, M.C.; Alvarez, M.V.; Izquierdo, M.C.

1990-01-01

The radiosensitizing effect of five glyoxal derivatives on the survival of TC-SV40 cells has been measured, under aerobic and hypoxic conditions. A toxicity study was previously performed in order to use nontoxic concentrations. The OER for the TC-SV40 cells was 2.74. None of the glyoxylic compounds showed radiosensitizing activity under aerobic conditions while in hypoxia their radiosensitizing factors decreased in the order phenylglyoxylic acid (1.68 at 8 x 10(-3) mole dm-3) greater than phenylglyoxal (1.55 at 5 x 10(-6) mole dm-3) greater than 2-2' furil (1.48 at 5 x 10(-5) mole dm-3) greater than glyoxylic acid (1.39 at 1 x 10(-3) mole dm-3) greater than glyoxal (1.30 at 5 x 10(-5) mole dm-3). The dose-modifying factors were also determined at two equimolar concentrations 5 x 10(-5) and 5 x 10(-6) mole dm-3. A concentration effect was noticed for all the compounds although their relative radiosensitizing activity kept, independently of the concentration, the same order noted above. Glyoxals with aromatic or heterocyclic rings exert a greater radiosensitization than the others. The acidic compounds have less radiosensitizing activity than their aldehydic counterparts. Interaction of these glyoxals with NPSH cellular groups was tested and the low degree of inhibition shows that this mechanism would contribute very little, if any, to the radiosensitization effect

Targeting hypoxic microenvironment of pancreatic xenografts with the hypoxia-activated prodrug TH-302.

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Lohse, Ines; Rasowski, Joanna; Cao, Pinjiang; Pintilie, Melania; Do, Trevor; Tsao, Ming-Sound; Hill, Richard P; Hedley, David W

2016-06-07

Previous reports have suggested that the hypoxic microenvironment provides a niche that supports tumor stem cells, and that this might explain clinical observations linking hypoxia to metastasis. To test this, we examined the effects of a hypoxia-activated prodrug, TH-302, on the tumor-initiating cell (TIC) frequency of patient-derived pancreatic xenografts (PDX).The frequencies of TIC, measured by limiting dilution assay, varied widely in 11 PDX models, and were correlated with rapid growth but not with the levels of hypoxia. Treatment with either TH-302 or ionizing radiation (IR), to target hypoxic and well-oxygenated regions, respectively, reduced TIC frequency, and the combination of TH-302 and IR was much more effective in all models tested. The combination was also more effective than TH-302 or IR alone controlling tumor growth, particularly treating the more rapidly-growing/hypoxic models. These findings support the clinical utility of hypoxia targeting in combination with radiotherapy to treat pancreatic cancers, but do not provide strong evidence for a hypoxic stem cell niche.

Basic fibroblast growth factor enhances cell proliferation in the dentate gyrus of neonatal rats following hypoxic-ischemic brain damage.

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Zhu, Huan; Qiao, Lixing; Sun, Yao; Yin, Liping; Huang, Li; Jiang, Li; Li, Jiaqing

2018-04-23

Perinatal hypoxic-ischemic insult is considered a major contributor to child mortality and morbidity and leads to neurological deficits in newborn infants. There has been a lack of promising neurotherapeutic interventions for hypoxic-ischemic brain damage (HIBD) for clinical application in infants. The present study aimed to investigate the correlation between neurogenesis and basic fibroblast growth factor (bFGF) in the hippocampal dentate gyrus (DG) region in neonatal rats following HIBD. Cell proliferation was examined by detecting BrdU signals, and the role of bFGF in cell proliferation in the DG region following neonatal HIBD was investigated. Cell proliferation was induced by HIBD in the hippocampal DG of neonatal rats. Furthermore, bFGF gene expression was upregulated in the hippocampus in neonatal rats, particularly between 7 and 14 days after HIBD. Moreover, intraperitoneal injection of exogenous bFGF enhanced cell proliferation in the hippocampal DG following neonatal HIBD. Taken together, these data indicate that cell proliferation in the DG could be induced by neonatal HIBD, and bFGF promotes proliferation following neonatal HIBD. Copyright © 2018 Elsevier B.V. All rights reserved.

The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood brain barrier disruption

International Nuclear Information System (INIS)

Yasui, Hironobu; Asanuma, Taketoshi; Kino, Junichi; Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Kubota, Nobuo; Kuwabara, Mikinori; Inanami, Osamu

2013-01-01

Glioblastoma is one of the intractable cancers and is highly resistant to ionizing radiation. This radioresistance is partly due to the presence of a hypoxic region which is widely found in advanced malignant gliomas. In the present study, we evaluated the effectiveness of the hypoxic cell sensitizer doranidazole (PR-350) using the C6 rat glioblastoma model, focusing on the status of blood brain barrier (BBB). Reproductive cell death in the rat C6 glioma cell line was determined by means of clonogenic assay. An intracranial C6 glioma model was established for the in vivo experiments. To investigate the status of the BBB in C6 glioma bearing brain, we performed the Evans blue extravasation test. Autoradiography with [ 14 C]-doranidazole was performed to examine the distribution of doranidazole in the glioma tumor. T2-weighted MRI was employed to examine the effects of X-irradiation and/or doranidazole on tumor growth. Doranidazole significantly enhanced radiation-induced reproductive cell death in vitro under hypoxia, but not under normoxia. The BBB in C6-bearing brain was completely disrupted and [ 14 C]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas. Our results revealed that BBB disruption in glioma enables BBB-impermeable radiosensitizers to penetrate and distribute in the target region. This study is the first to propose that in malignant glioma the administration of hydrophilic hypoxic radiosensitizers could be a potent strategy for improving the clinical outcome of radiotherapy without side effects

KIH-802: 2-nitroimidazole-1-acetohydroxamate as a hypoxic cell radiosensitizer

International Nuclear Information System (INIS)

Hori, H.; Murayama, C.; Mori, T.; Shibamoto, Y.; Abe, M.; Onoyama, Y.; Inayama, S.

1989-01-01

We have identified potassium 2-nitroimidazole-1-acetohydroxamate (KIH-802) as a hypoxic cell radiosensitizer potentially superior to Miso. The water-soluble acetohydroxamates of 2-nitroimidazole (KIH-802; free acid 801) and 4-nitroimidazole (KIH-852) were designed, synthesized, and evaluated by in vitro and in vivo screening against EMT6 cells. Enhancement ratios of KIH-802 and 801 were 1.92 and 1.68, respectively, compared with 1.58 for MISO all at 1 mM. These acetohydroxamates are also expected to be more effective in vitro than SR-2508 based on our previous experiments. In vivo ERs of KIH-802, 801, and 852 were 1.75, 1.50, and 1.35, respectively, compared with 1.57 for MISO all at the same dose of 200 mg/kg. The data clearly show that the addition of an acetohydroxamic acid moiety to the 2-nitroimidazole skeleton can enhance radiosensitizing ability

Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1α under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells

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Dong Wei

2013-04-01

Full Text Available Digoxin, an inhibitor of Na+/K+ ATPase, has been used in the treatment of heart-related diseases (such as congestive heart failure and atrial arrhythmia for decades. Recently, it was reported that digoxin is also an effective HIF-1α inhibitor. We investigated whether digoxin could suppress tumor cell growth through HIF-1α in non-small cell lung cancer cells (A549 cells under hypoxic conditions. An MTT assay was used to measure cell viability. RT-PCR and western blotting were performed to analyze the mRNA and protein expression of VEGF, NDRG1, and HIF-1α. HIF-1α nuclear translocation was then determined by EMSA. Digoxin was found to inhibit the proliferation of A549 cells under hypoxic conditions. Our results showed that hypoxia led to the upregulation of VEGF, NDRG1, and HIF-1α both at the mRNA and protein levels. We also found that the hypoxia-induced overexpression of VEGF, NDRG1, and HIF-1α was suppressed by digoxin in a concentration-dependent manner. As expected, our EMSA results demonstrated that under hypoxic conditions HIF-1α nuclear translocation was also markedly reduced by digoxin in a concentration-dependent manner. Our results suggest that digoxin downregulated hypoxia-induced overexpression of VEGF and NDRG1 at the transcriptional level probably through the inhibition of HIF-1α synthesis in A549 cells.

Azobenzene-caged sulforhodamine dyes: a novel class of ‘turn-on’ reactive probes for hypoxic tumor cell imaging

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Chevalier, Arnaud; Piao, Wen; Hanaoka, Kenjiro; Nagano, Tetsuo; Renard, Pierre-Yves; Romieu, Anthony

2015-12-01

New sulforhodamine-based fluorescent ‘turn-on’ probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble ‘azobenzene-caged’ fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH 2 ) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH 2 . This valuable fluorogenic response was obtained for the three ‘smart’ probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH 2 -Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

Compliance and toxicity of the hypoxic radiosensitizer nimorazole in the treatment of patients with head and neck squamous cell carcinoma (HNSCC)

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Metwally, Mohamed Ahmed Hassan; Frederiksen, Katrine Diemer; Overgaard, Jens

2014-01-01

Abstract Purpose. To evaluate the compliance and toxicity of the hypoxic radiosensitizer nimorazole in head and neck cancer patients. Methods. A retrospective study of patients with head and neck squamous cell carcinoma (HNSCC), treated in Denmark between 1990 and 2013. All patients treated...... with radical radiotherapy (± chemotherapy) [66-70 Gy; 33-35 fractions; 2 Gy/fraction; 5-6 fractions/week] concomitant with the hypoxic radiosensitizer nimorazole. Nimorazole was administered as oral tablets in doses of approximately 1.2 g/m(2) body surface area in connection with the first of each daily...

Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

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Copple, Bryan L; Bustamante, Juan J; Welch, Timothy P; Kim, Nam Deuk; Moon, Jeon-Ok

2009-08-01

During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.

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

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

2013-01-01

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

Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

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Emara Marwan

2010-09-01

Full Text Available Abstract Background Cytoglobin (Cygb and neuroglobin (Ngb are recently identified globin molecules that are expressed in vertebrate tissues. Upregulation of Cygb and Ngb under hypoxic and/or ischemic conditions in vitro and in vivo increases cell survival, suggesting possible protective roles through prevention of oxidative damage. We have previously shown that Ngb is expressed in human glioblastoma multiforme (GBM cell lines, and that expression of its transcript and protein can be significantly increased after exposure to physiologically relevant levels of hypoxia. In this study, we extended this work to determine whether Cygb is also expressed in GBM cells, and whether its expression is enhanced under hypoxic conditions. We also compared Cygb and Ngb expression in human primary tumor specimens, including brain tumors, as well as in human normal tissues. Immunoreactivity of carbonic anhydrase IX (CA IX, a hypoxia-inducible metalloenzyme that catalyzes the hydration of CO2 to bicarbonate, was used as an endogenous marker of hypoxia. Results Cygb transcript and protein were expressed in human GBM cells, and this expression was significantly increased in most cells following 48 h incubation under hypoxia. We also showed that Cygb and Ngb are expressed in both normal tissues and human primary cancers, including GBM. Among normal tissues, Cygb and Ngb expression was restricted to distinct cell types and was especially prominent in ductal cells. Additionally, certain normal organs (e.g. stomach fundus, small bowel showed distinct regional co-localization of Ngb, Cygb and CA IX. In most tumors, Ngb immunoreactivity was significantly greater than that of Cygb. In keeping with previous in vitro results, tumor regions that were positively stained for CA IX were also positive for Ngb and Cygb, suggesting that hypoxic upregulation of Ngb and Cygb also occurs in vivo. Conclusions Our finding of hypoxic up-regulation of Cygb/Ngb in GBM cell lines and human

Doranidazole (PR-350), a hypoxic cell radiosensitizer, radiosensitizes human lung tumors (RERF-LC- AI) and causes changes in tumor oxygenation

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Kubota, N.; Griffin, R.J.; Williams, B.W.; Song, C.W.; Yahiro, T.

2003-01-01

Full text: We previously have reported the radiosensitizing capability of Doranidazole (PR-350) on SCCVII cells and tumors (Puerto Rico, 2001). In the present study, we have investigated the efficacy of PR-350 as a hypoxic cell radiosensitizer using human lung cancer cells (RERF-LC-AI) in vitro and also RERF-LC-AI tumors grown s.c. in Balb/c nude mice. Using the micronucleus assay method, we determined the effect of PR-350 on the response of RERF-LC-AI cells to radiation under hypoxic conditions and enhancement ratios (ER) of 1.45∼2.26 were obtained. The in vivo radiosensitizing effect was studied by irradiating RERF-LC-AI tumors with 15 Gy at 20 min. after i.v. injection of PR-350 (200mg/kg) and measuring the tumor growth delay. Significant growth delay occurred after i.v. injection of PR-350 before irradiation compared to radiation alone. We measured tumor pO 2 at 3, 7 and 14 days after treatment using an Eppendorf pO 2 histograph. The frequency of pO 2 values 2 in tumors treated with radiation plus PR-350 were higher than that in tumors treated with radiation plus saline. These data suggest that the O 2 consumption in tumors treated with radiation plus PR-350 was less than that in tumors treated with radiation plus saline due to greater drug and radiation-induced cell death. This hypothesis is supported by the fact that the tumor size in the combined treatment group was smaller than in radiation alone. These results suggest that PR-350 may improve the response of tumors to radiotherapy not only by increasing the radiosensitivity of hypoxic cells but also by improving tumor oxygenation over many days during fractionated radiotherapy

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

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

2017-06-01

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

Hypoxia-inducible factor 1-alpha up-regulates the expression of phospholipase D2 in colon cancer cells under hypoxic conditions.

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Liu, Maoxi; Du, Kunli; Fu, Zhongxue; Zhang, Shouru; Wu, Xingye

2015-01-01

Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that phospholipase D2 (PLD2) plays significant roles in cancer progression. In this study, correlation between the expression of PLD2 and the change in the protein level of hypoxia-inducible factor 1-alpha (HIF1-α) was studied. Thirty human colon cancer tissues were examined for the expression of HIF1-α and PLD2 protein, and mRNA levels. SW480 and SW620 cells were exposed to normoxia (20 %) or hypoxia (Hypoxic stress induced PLD2 mRNA and protein expression in SW480 and SW620 cells. Cells transfected with HIF1-α siRNA showed attenuation of hypoxia stress-induced PLD2 expression. In vivo growth decreased in response to HIF1-α and PLD2 inhibition. These results suggest that PLD2 expression in colon cancer cells is up-regulated via HIF1-α in response to hypoxic stress and underscores the crucial role of HIF1-α-induced PLD2 in tumor growth.

Hypoxic Culture Promotes Dopaminergic-Neuronal Differentiation of Nasal Olfactory Mucosa Mesenchymal Stem Cells via Upregulation of Hypoxia-Inducible Factor-1α.

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Zhuo, Yi; Wang, Lei; Ge, Lite; Li, Xuan; Duan, Da; Teng, Xiaohua; Jiang, Miao; Liu, Kai; Yuan, Ting; Wu, Pei; Wang, Hao; Deng, Yujia; Xie, Huali; Chen, Ping; Xia, Ying; Lu, Ming

2017-08-01

Olfactory mucosa mesenchymal stem cells (OM-MSCs) display significant clonogenic activity and may be easily propagated for Parkinson's disease therapies. Methods of inducing OM-MSCs to differentiate into dopaminergic (DAergic) neurons using olfactory ensheathing cells (OECs) are thus an attractive topic of research. We designed a hypoxic induction protocol to generate DAergic neurons from OM-MSCs using a physiological oxygen (O 2 ) level of 3% and OEC-conditioned medium (OCM; HI group). The normal induction (NI) group was cultured in O 2 at ambient air level (21%). The role of hypoxia-inducible factor-1α (HIF-1α) in the differentiation of OM-MSCs under hypoxia was investigated by treating cells with an HIF-1α inhibitor before induction (HIR group). The proportions of β-tubulin- and tyrosine hydroxylase (TH)-positive cells were significantly increased in the HI group compared with the NI and HIR groups, as shown by immunocytochemistry and Western blotting. Furthermore, the level of dopamine was significantly increased in the HI group. A slow outward potassium current was recorded in differentiated cells after 21 d of induction using whole-cell voltage-clamp tests. A hypoxic environment thus promotes OM-MSCs to differentiate into DAergic neurons by increasing the expression of HIF-1α and by activating downstream target gene TH. This study indicated that OCM under hypoxic conditions could significantly upregulate key transcriptional factors involved in the development of DAergic neurons from OM-MSCs, mediated by HIF-1α. Hypoxia promotes DAergic neuronal differentiation of OM-MSCs, and HIF-1α may play an important role in hypoxia-inducible pathways during DAergic lineage specification and differentiation in vitro.

Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility.

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Sada, Masafumi; Ohuchida, Kenoki; Horioka, Kohei; Okumura, Takashi; Moriyama, Taiki; Miyasaka, Yoshihiro; Ohtsuka, Takao; Mizumoto, Kazuhiro; Oda, Yoshinao; Nakamura, Masafumi

2016-03-28

Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

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

2012-02-01

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

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

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Thiersch, Markus; Lange, Christina; Joly, Sandrine; Heynen, Severin; Le, Yun Zheng; Samardzija, Marijana; Grimm, Christian

2009-06-01

Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality

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Jiang, Yanyan; Verbiest, Tom; Devery, Aoife M.; Bokobza, Sivan M.; Weber, Anika M.; Leszczynska, Katarzyna B.; Hammond, Ester M.; Ryan, Anderson J., E-mail: anderson.ryan@oncology.ox.ac.uk

2016-06-01

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models. Methods and Materials: NSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O{sub 2}) or hypoxic (1% O{sub 2}) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, daily for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth. Results: In vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O{sub 2}). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors. Conclusions: Our data suggest that hypoxia potentiates the radiation-sensitizing effects of

Colony, hanging drop, and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines.

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Matak, Damian; Brodaczewska, Klaudia K; Lipiec, Monika; Szymanski, Łukasz; Szczylik, Cezary; Czarnecka, Anna M

2017-08-01

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.

Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury by suppressing apoptosis

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De-An Zhao

Full Text Available Abstract Background and objectives: Isoflurane is halogenated volatile ether used for inhalational anesthesia. It is widely used in clinics as an inhalational anesthetic. Neonatal hypoxic ischemia injury ensues in the immature brain that results in delayed cell death via excitotoxicity and oxidative stress. Isoflurane has shown neuroprotective properties that make a beneficial basis of using isoflurane in both cell culture and animal models, including various models of brain injury. We aimed to determine the neuroprotective effect of isoflurane on hypoxic brain injury and elucidated the underlying mechanism. Methods: A hippocampal slice, in artificial cerebrospinal fluid with glucose and oxygen deprivation, was used as an in vitro model for brain hypoxia. The orthodromic population spike and hypoxic injury potential were recorded in the CA1 and CA3 regions. Amino acid neurotransmitters concentration in perfusion solution of hippocampal slices was measured. Results: Isoflurane treatment caused delayed elimination of population spike and improved the recovery of population spike; decreased frequency of hypoxic injury potential, postponed the onset of hypoxic injury potential and increased the duration of hypoxic injury potential. Isoflurane treatment also decreased the hypoxia-induced release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but the levels of γ-aminobutyric acid were elevated. Morphological studies showed that isoflurane treatment attenuated edema of pyramid neurons in the CA1 region. It also reduced apoptosis as evident by lowered expression of caspase-3 and PARP genes. Conclusions: Isoflurane showed a neuro-protective effect on hippocampal neuron injury induced by hypoxia through suppression of apoptosis.

A voxel-based multiscale model to simulate the radiation response of hypoxic tumors.

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Espinoza, I; Peschke, P; Karger, C P

2015-01-01

In radiotherapy, it is important to predict the response of tumors to irradiation prior to the treatment. This is especially important for hypoxic tumors, which are known to be highly radioresistant. Mathematical modeling based on the dose distribution, biological parameters, and medical images may help to improve this prediction and to optimize the treatment plan. A voxel-based multiscale tumor response model for simulating the radiation response of hypoxic tumors was developed. It considers viable and dead tumor cells, capillary and normal cells, as well as the most relevant biological processes such as (i) proliferation of tumor cells, (ii) hypoxia-induced angiogenesis, (iii) spatial exchange of cells leading to tumor growth, (iv) oxygen-dependent cell survival after irradiation, (v) resorption of dead cells, and (vi) spatial exchange of cells leading to tumor shrinkage. Oxygenation is described on a microscopic scale using a previously published tumor oxygenation model, which calculates the oxygen distribution for each voxel using the vascular fraction as the most important input parameter. To demonstrate the capabilities of the model, the dependence of the oxygen distribution on tumor growth and radiation-induced shrinkage is investigated. In addition, the impact of three different reoxygenation processes is compared and tumor control probability (TCP) curves for a squamous cells carcinoma of the head and neck (HNSSC) are simulated under normoxic and hypoxic conditions. The model describes the spatiotemporal behavior of the tumor on three different scales: (i) on the macroscopic scale, it describes tumor growth and shrinkage during radiation treatment, (ii) on a mesoscopic scale, it provides the cell density and vascular fraction for each voxel, and (iii) on the microscopic scale, the oxygen distribution may be obtained in terms of oxygen histograms. With increasing tumor size, the simulated tumors develop a hypoxic core. Within the model, tumor shrinkage was

A voxel-based multiscale model to simulate the radiation response of hypoxic tumors

International Nuclear Information System (INIS)

Espinoza, I.; Peschke, P.; Karger, C. P.

2015-01-01

Purpose: In radiotherapy, it is important to predict the response of tumors to irradiation prior to the treatment. This is especially important for hypoxic tumors, which are known to be highly radioresistant. Mathematical modeling based on the dose distribution, biological parameters, and medical images may help to improve this prediction and to optimize the treatment plan. Methods: A voxel-based multiscale tumor response model for simulating the radiation response of hypoxic tumors was developed. It considers viable and dead tumor cells, capillary and normal cells, as well as the most relevant biological processes such as (i) proliferation of tumor cells, (ii) hypoxia-induced angiogenesis, (iii) spatial exchange of cells leading to tumor growth, (iv) oxygen-dependent cell survival after irradiation, (v) resorption of dead cells, and (vi) spatial exchange of cells leading to tumor shrinkage. Oxygenation is described on a microscopic scale using a previously published tumor oxygenation model, which calculates the oxygen distribution for each voxel using the vascular fraction as the most important input parameter. To demonstrate the capabilities of the model, the dependence of the oxygen distribution on tumor growth and radiation-induced shrinkage is investigated. In addition, the impact of three different reoxygenation processes is compared and tumor control probability (TCP) curves for a squamous cells carcinoma of the head and neck (HNSSC) are simulated under normoxic and hypoxic conditions. Results: The model describes the spatiotemporal behavior of the tumor on three different scales: (i) on the macroscopic scale, it describes tumor growth and shrinkage during radiation treatment, (ii) on a mesoscopic scale, it provides the cell density and vascular fraction for each voxel, and (iii) on the microscopic scale, the oxygen distribution may be obtained in terms of oxygen histograms. With increasing tumor size, the simulated tumors develop a hypoxic core. Within the

PERK/eIF2alpha signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS

NARCIS (Netherlands)

Rouschop, K.M.; Dubois, L.J.; Keulers, T.G.; Beucken, T. van den; Lambin, P.; Bussink, J.; Kogel, A.J. van der; Koritzinsky, M.; Wouters, B.G.

2013-01-01

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic

MicroRNA-195 induced apoptosis in hypoxic chondrocytes by targeting hypoxia-inducible factor 1 alpha.

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Bai, R; Zhao, A-Q; Zhao, Z-Q; Liu, W-L; Jian, D-M

2015-02-01

The chondrocytes, the resident cells of cartilage, are maintained and take effects in the whole life upon chronic hypoxic exposure, which hypoxia-inducible factor 1 alpha (HIF-1α) play pivotal roles in response to. Dysregulation of some microRNA (miRNAs) have also been identified to be involved in hypoxia-related physiologic and pathophysiologic responses in some tissues or cell lines. However, the mechanism of miRNAs reponse to hypoxia remain largely unknown in chondrocytes, including the microRNA-195 (miR-195). AIM To investigate the effects of microRNAs (miRNAs) and hypoxia-inducible factor 1 alpha (HIF-1α) on chondrocytes in physiologic environment. We compared the expression of miR-195 and HIF-1α mRNA on hypoxia with that on normoxia in ATDC 5 cells by qRT-PCR. Further experiments was performed to confirmed the relationships of miR-195 and HIF-1α by bioinformatics analysis and dual reporter gene assay. we also assessed the effect of miR-195 on apoptosis in hypoxic ATDC 5 cells by transfect with miR-195 mimics. It was found the downregulated miR-195 and upregulated HIF-1α were present in hypoxic ATDC 5 cells. miR-195 negatively regulated HIF-1α by targeting its 3'-untranslated region. Moreover, the founding indicated miR-195 greatly increased apoptosis and downregulated HIF-1α mRNA occurred simultaneously in hypoxic chondrocytes. We concluded that miR-195 induced apoptosis in hypoxic chondrocytes by directly targeting HIF-1α.

A low protein diet increases the hypoxic tolerance in Drosophila.

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Paul Vigne

2006-12-01

Full Text Available Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O(2 and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses.

Long noncoding RNA HOTAIR, a hypoxia-inducible factor-1α activated driver of malignancy, enhances hypoxic cancer cell proliferation, migration, and invasion in non-small cell lung cancer.

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Zhou, Chunxia; Ye, Lincai; Jiang, Chuan; Bai, Jie; Chi, Yongbin; Zhang, Haibo

2015-12-01

Despite the fact that great advances have been made in the management of non-small cell lung cancer (NSCLC), the prognosis of advanced NSCLC remains very poor. HOX transcript antisense intergenic RNA (HOTAIR) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in the progression of a variety of carcinomas and acts as a negative prognostic biomarker. Yet, little is known about the effect of HOTAIR in the hypoxic microenvironment of NSCLC. The expression and promoter activity of HOTAIR were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay. The function of the hypoxia-inducible factor-1α (HIF-1α) binding site to hypoxia-responsive elements (HREs) in the HOTAIR promoter region was tested by luciferase reporter assay with nucleotide substitutions. The binding of HIF-1α to the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). The effect of HIF-1α suppression by small interference RNA or YC-1 on HOTAIR expression was also determined. In the present study, we demonstrated that HOTAIR was upregulated by hypoxia in NSCLC cells. HOTAIR is a direct target of HIF-1α through interaction with putative HREs in the upstream region of HOTAIR in NSCLC cells. Furthermore, HIF-1α knockdown or inhibition could prevent HOTAIR upregulation under hypoxic conditions. Under hypoxic conditions, HOTAIR enhanced cancer cell proliferation, migration, and invasion. These data suggested that suppression of HOTAIR upon hypoxia of NSCLC could be a novel therapeutic strategy.

The feasibility of a targeted ultrasound contrast agent carrying genes and cell-penetrating peptides to hypoxic HUVEC

International Nuclear Information System (INIS)

Tian Ju; Wang Zhigang; Ren Jianli; Zhang Qingfeng; Liu Li

2012-01-01

Objective: To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides (CPP) and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells (HUVEC). Methods: Anti-P-selectin targeted ultrasound contrast agent carrying a green fluorescent protein gene (pEGFP-N1) and CPP was prepared by mechanical vibration and carbodiimide techniques. The appearance, distribution, concentration and diameter of the ultrasound contrast agent were measured. The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy (CLSM). The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry. HUVEC were cultured in vitro and hypoxic HUVEC were prepared using hydrogen peroxide (H 2 O 2 ). Hypoxic HUVEC were randomly assigned targeted ultrasound contrast agents and non-targeted ultrasound contrast agents for transfection. The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry. T-test and linear correlation analysis were used for statistical analysis. Results: The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was (2.15 ±0.36) μm and the concentration was (1.58 ± 0.23) × 10 7 /ml.The results of CLSM showed that gene and CPP were distributed on the shell of the agent. The gene encapsulation efficiency was 28% (y=0.932x-0.09, r=0.993, P<0.05), and the CPP encapsulation efficiency was 25% (y=5.875x-0.81, r=0.987, P<0.05). EGFP expression was observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted ultrasound contrast agents. The average transfection efficiencies of targeted ultrasound contrast agents and non-targeted ultrasound contrast agents were (18.74 ± 0.47) % and (15.34 ± 0.22) % after 24 h (t=10.923, P<0.001). Conclusions: The in vitro studies

Urothelial carcinoma associated 1 is a hypoxia-inducible factor-1α-targeted long noncoding RNA that enhances hypoxic bladder cancer cell proliferation, migration, and invasion.

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Xue, Mei; Li, Xu; Li, Zhengkun; Chen, Wei

2014-07-01

Urothelial carcinoma associated 1 (UCA1) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in bladder cancer progression and acts as a diagnostic biomarker for bladder carcinoma. Here, we studied the expression and function of lncRNA-UCA1 in the hypoxic microenvironment of bladder cancer. The expression and transcriptional activity of lncRNA-UCA1 were measured by quantitative real-time polymerase chain reaction and luciferase assays. Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry. Cell migration and invasion were detected by wound healing, migration, and invasion assays. The binding of hypoxia-inducible factor-1α (HIF-1α) to hypoxia response elements (HREs) in the lncRNA-UCA1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. HRE mutations were generated by using a site-directed mutagenesis kit, and HIF-1α knockdown was mediated by small interfering RNA. The effect of HIF-1α inhibition by YC-1 on lncRNA-UCA1 expression was also examined. LncRNA-UCA1 was upregulated by hypoxia in bladder cancer cells. Under hypoxic conditions, lncRNA-UCA1 upregulation increased cell proliferation, migration, and invasion and inhibited apoptosis. The underlying mechanism of hypoxia-upregulated lncRNA-UCA1 expression was that HIF-1α specifically bound to HREs in the lncRNA-UCA1 promoter. Furthermore, HIF-1α knockdown or inhibition could prevent lncRNA-UCA1 upregulation under hypoxia. These findings revealed the mechanism of lncRNA-UCA1 upregulation in hypoxic bladder cancer cells and suggested that effective blocking of lncRNA-UCA1 expression in the hypoxic microenvironment of bladder cancer could be a novel therapeutic strategy.

Reaction between nitracrine and glutathione: implications for hypoxic cell radiosensitization and cytotoxicity

International Nuclear Information System (INIS)

Wilson, W.R.; Anderson, R.F.

1989-01-01

Nitracrine (NC) is an electron affinic DNA intercalating agent and a potent hypoxia-selective cytotoxin and radiosensitizer in cell culture. Although NC is too cytotoxic and too rapidly metabolized to provide hypoxic cell radiosensitization in tumors, it is of mechanistic interest as an example of a DNA affinic radiosensitizer. We have observed a rapid chemical reaction between NC and reduced glutathione (GSH), which suggests that the observed potent in vitro cytotoxicity and radiosensitization might be dependent on thiol depletion by the large extracellular reservoir of drug. However, no GSH depletion was observed under conditions providing radiosensitization or rapid cell killing, and prior depletion of GSH by buthionine sulphoximine had no effect on cytotoxicity or formation of macromolecular adducts. Further, the intracellular reaction of NC with GSH is slower than predicted on the basis of the measured second order rate constant and the total intracellular concentrations of both species. The results are consistent with a role for DNA binding in protecting NC from reaction with GSH, and in improving the efficiency with which reduced electrophilic metabolites react with DNA in preference to GSH

[Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury by suppressing apoptosis].

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Zhao, De-An; Bi, Ling-Yun; Huang, Qian; Zhang, Fang-Min; Han, Zi-Ming

Isoflurane is halogenated volatile ether used for inhalational anesthesia. It is widely used in clinics as an inhalational anesthetic. Neonatal hypoxic ischemia injury ensues in the immature brain that results in delayed cell death via excitotoxicity and oxidative stress. Isoflurane has shown neuroprotective properties that make a beneficial basis of using isoflurane in both cell culture and animal models, including various models of brain injury. We aimed to determine the neuroprotective effect of isoflurane on hypoxic brain injury and elucidated the underlying mechanism. A hippocampal slice, in artificial cerebrospinal fluid with glucose and oxygen deprivation, was used as an in vitro model for brain hypoxia. The orthodromic population spike and hypoxic injury potential were recorded in the CA1 and CA3 regions. Amino acid neurotransmitters concentration in perfusion solution of hippocampal slices was measured. Isoflurane treatment caused delayed elimination of population spike and improved the recovery of population spike; decreased frequency of hypoxic injury potential, postponed the onset of hypoxic injury potential and increased the duration of hypoxic injury potential. Isoflurane treatment also decreased the hypoxia-induced release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but the levels of γ-aminobutyric acid were elevated. Morphological studies showed that isoflurane treatment attenuated edema of pyramid neurons in the CA1 region. It also reduced apoptosis as evident by lowered expression of caspase-3 and PARP genes. Isoflurane showed a neuro-protective effect on hippocampal neuron injury induced by hypoxia through suppression of apoptosis. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury by suppressing apoptosis.

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Zhao, De-An; Bi, Ling-Yun; Huang, Qian; Zhang, Fang-Min; Han, Zi-Ming

Isoflurane is halogenated volatile ether used for inhalational anesthesia. It is widely used in clinics as an inhalational anesthetic. Neonatal hypoxic ischemia injury ensues in the immature brain that results in delayed cell death via excitotoxicity and oxidative stress. Isoflurane has shown neuroprotective properties that make a beneficial basis of using isoflurane in both cell culture and animal models, including various models of brain injury. We aimed to determine the neuroprotective effect of isoflurane on hypoxic brain injury and elucidated the underlying mechanism. A hippocampal slice, in artificial cerebrospinal fluid with glucose and oxygen deprivation, was used as an in vitro model for brain hypoxia. The orthodromic population spike and hypoxic injury potential were recorded in the CA1 and CA3 regions. Amino acid neurotransmitters concentration in perfusion solution of hippocampal slices was measured. Isoflurane treatment caused delayed elimination of population spike and improved the recovery of population spike; decreased frequency of hypoxic injury potential, postponed the onset of hypoxic injury potential and increased the duration of hypoxic injury potential. Isoflurane treatment also decreased the hypoxia-induced release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but the levels of γ-aminobutyric acid were elevated. Morphological studies showed that isoflurane treatment attenuated edema of pyramid neurons in the CA1 region. It also reduced apoptosis as evident by lowered expression of caspase-3 and PARP genes. Isoflurane showed a neuro-protective effect on hippocampal neuron injury induced by hypoxia through suppression of apoptosis. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

Hypoxic preconditioning induces neuroprotective stanniocalcin-1 in brain via IL-6 signaling

DEFF Research Database (Denmark)

Westberg, Johan A; Serlachius, Martina; Lankila, Petri

2007-01-01

BACKGROUND AND PURPOSE: Exposure of animals for a few hours to moderate hypoxia confers relative protection against subsequent ischemic brain damage. This phenomenon, known as hypoxic preconditioning, depends on new RNA and protein synthesis, but its molecular mechanisms are poorly understood...... originally reported expression of mammalian STC-1 in brain neurons and showed that STC-1 guards neurons against hypercalcemic and hypoxic damage. METHODS: We treated neural Paju cells with IL-6 and measured the induction of STC-1 mRNA. In addition, we quantified the effect of hypoxic preconditioning on Stc-1...... mRNA levels in brains of wild-type and IL-6 deficient mice. Furthermore, we monitored the Stc-1 response in brains of wild-type and transgenic mice, overexpressing IL-6 in the astroglia, before and after induced brain injury. RESULTS: Hypoxic preconditioning induced an upregulated expression of Stc...

SU-E-T-146: Effects of Uncertainties of Radiation Sensitivity of Biological Modelling for Treatment Planning

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Oita, M [Department of Radiological Technology, Graduate School of Health Sciences, Okayama University (Japan); Department of Life System, Institute of Technology and Science, Graduate School, The Tokushima University (Japan); Uto, Y; Hori, H [Department of Life System, Institute of Technology and Science, Graduate School, The Tokushima University (Japan); Tominaga, M [Department of Radiological Technology, Institute of Health Biosciences, Graduate School, The Tokushima University (Japan); Sasaki, M [Department of Radiology, Tokushima University Hospital (Japan)

2014-06-01

Purpose: The aim of this study was to evaluate the distribution of uncertainty of cell survival by radiation, and assesses the usefulness of stochastic biological model applying for gaussian distribution. Methods: For single cell experiments, exponentially growing cells were harvested from the standard cell culture dishes by trypsinization, and suspended in test tubes containing 1 ml of MEM(2x10{sup 6} cells/ml). The hypoxic cultures were treated with 95% N{sub 2}−5% CO{sub 2} gas for 30 minutes. In vitro radiosensitization was also measured in EMT6/KU single cells to add radiosensitizer under hypoxic conditions. X-ray irradiation was carried out by using an Xray unit (Hitachi X-ray unit, model MBR-1505R3) with 0.5 mm Al/1.0 mm Cu filter, 150 kV, 4 Gy/min). In vitro assay, cells on the dish were irradiated with 1 Gy to 24 Gy, respectively. After irradiation, colony formation assays were performed. Variations of biological parameters were investigated at standard cell culture(n=16), hypoxic cell culture(n=45) and hypoxic cell culture(n=21) with radiosensitizers, respectively. The data were obtained by separate schedule to take account for the variation of radiation sensitivity of cell cycle. Results: At standard cell culture, hypoxic cell culture and hypoxic cell culture with radiosensitizers, median and standard deviation of alpha/beta ratio were 37.1±73.4 Gy, 9.8±23.7 Gy, 20.7±21.9 Gy, respectively. Average and standard deviation of D{sub 50} were 2.5±2.5 Gy, 6.1±2.2 Gy, 3.6±1.3 Gy, respectively. Conclusion: In this study, we have challenged to apply these uncertainties of parameters for the biological model. The variation of alpha values, beta values, D{sub 50} as well as cell culture might have highly affected by probability of cell death. Further research is in progress for precise prediction of the cell death as well as tumor control probability for treatment planning.

SU-E-T-146: Effects of Uncertainties of Radiation Sensitivity of Biological Modelling for Treatment Planning

International Nuclear Information System (INIS)

Oita, M; Uto, Y; Hori, H; Tominaga, M; Sasaki, M

2014-01-01

Purpose: The aim of this study was to evaluate the distribution of uncertainty of cell survival by radiation, and assesses the usefulness of stochastic biological model applying for gaussian distribution. Methods: For single cell experiments, exponentially growing cells were harvested from the standard cell culture dishes by trypsinization, and suspended in test tubes containing 1 ml of MEM(2x10 6 cells/ml). The hypoxic cultures were treated with 95% N 2 −5% CO 2 gas for 30 minutes. In vitro radiosensitization was also measured in EMT6/KU single cells to add radiosensitizer under hypoxic conditions. X-ray irradiation was carried out by using an Xray unit (Hitachi X-ray unit, model MBR-1505R3) with 0.5 mm Al/1.0 mm Cu filter, 150 kV, 4 Gy/min). In vitro assay, cells on the dish were irradiated with 1 Gy to 24 Gy, respectively. After irradiation, colony formation assays were performed. Variations of biological parameters were investigated at standard cell culture(n=16), hypoxic cell culture(n=45) and hypoxic cell culture(n=21) with radiosensitizers, respectively. The data were obtained by separate schedule to take account for the variation of radiation sensitivity of cell cycle. Results: At standard cell culture, hypoxic cell culture and hypoxic cell culture with radiosensitizers, median and standard deviation of alpha/beta ratio were 37.1±73.4 Gy, 9.8±23.7 Gy, 20.7±21.9 Gy, respectively. Average and standard deviation of D 50 were 2.5±2.5 Gy, 6.1±2.2 Gy, 3.6±1.3 Gy, respectively. Conclusion: In this study, we have challenged to apply these uncertainties of parameters for the biological model. The variation of alpha values, beta values, D 50 as well as cell culture might have highly affected by probability of cell death. Further research is in progress for precise prediction of the cell death as well as tumor control probability for treatment planning

Methylation-mediated silencing of miR-124 facilitates chondrogenesis by targeting NFATc1 under hypoxic conditions.

Science.gov (United States)

Gong, Ming; Liang, Tangzhao; Jin, Song; Dai, Xuejun; Zhou, Zhiyu; Gao, Manman; Huang, Sheng; Luo, Jiaquan; Zou, Lijin; Zou, Xuenong

2017-01-01

Chondrogenic differentiation of mesenchymal stem cells is regulated by many different pathways. Recent studies have established that hypoxia and epigenetic alterations potently affect expression of chondrogenesis marker genes. Sox9 is generally regarded as a master regulator of chondrogenesis and microRNA-124 (miRNA-124) regulates gene expression in murine bone marrow-derived mesenchymal stem cells. Therefore, in this study we investigated whether epigenetic regulation of miRNA-124 could affect the expression of Sox9 and thereby regulate chondrogenesis. A cell pellet culture model was used to induce chondrogenesis in C3H10T1/2 cells under hypoxic conditions (2% O 2 ) to determine the effects of hypoxia on miR-124 expression and DNA methylation. The expression of miR-124 was significantly downregulated under hypoxic conditions compared to normoxic conditions (21% O 2 ). The expression of chondrogenesis marker genes was significantly increased under hypoxic conditions. Bisulfite sequencing of the CpG islands in the promoter region of miR-124-3 showed that CpG methylation was significantly increased under hypoxic conditions. Treating the cells with the DNA demethylating agent 5'-AZA significantly increased miR-124 expression and decreased expression of markers of chondrogenesis. Overexpressing miR-124 under hypoxic conditions inhibited NFATc1 reporter activity. NFATc1 was shown to bind to the promoter region of Sox9. Taken together, our data provide evidence that miR-124 acts as an inhibitor of NFATc1. Under hypoxic conditions when miR-124 is downregulated by methylation of CpG islands in the promoter, NFATc1 can bind to the Sox9 promoter and induce the expression of Sox9 leading to chondrogenesis. These results support the role of epigenetic regulation in establishing and maintaining a chondrogenic phenotype.

Study on intraoperative radiotherapy applying hyperthermia together with radiation sensitizers for progressive local carcinoma

Energy Technology Data Exchange (ETDEWEB)

Abe, M; Takahashi, M; Ono, K; Hiraoka, M [Kyoto Univ. (Japan). Faculty of Medicine

1980-08-01

Intraoperative radiotherapy for gastric cancer, colonic cancer, pancreatic cancer, cancer of the biliary tract, prostatic carcinoma, cerebral tumor, tumor of soft tissues, and osteosarcoma and its clinical results were described. Basic and clinical studies on effects of both hyperthermia and radiation sensitizers to elevate radiation sensitivity were also described, because effects of intraoperative radiotherapy were raised by applying hyperthermia and hypoxic cell sensitizers.

The radiosensitizing effects of ornidazole in hypoxic mammalian tissue: an in vivo study

International Nuclear Information System (INIS)

Okkan, S.; Uzel, R.

1982-01-01

In this study the sensitizing effects of ornidazole is investigated in vivo. The selected test system is the acute killing effect of radiation within 4-6 days after abdominal irradiation ranging from 9 to 24 Gy, in groups of C 57 black mice. Ornidazole is given intraperitoneally in 500 mg/kg, 100 mg/kg, 20 mg/kg doses prior to irradiation of animals breathing air, oxygen or nitrogen. A decreae of LD 50 dose is observed from 24.39 +/- 5.66 to 16.38 +/- 1.86 and 18.04 +/- 2.48 Gy, respectively, in nitrogen breathing animals. No sensitizing effect was observed in doses of 20 mg/kg. Enhancement Ratio (ER) was found to be 1.48 +/- 0.25 and 1.35 +/- 0.27; relative sensitizing efficiency (RSE) was 40% and 29% respectively. No sensitizing effect was observed in animals irradiated in oxic conditions. These results showed that ornidazole (Ro-7-0207) has a sensitizing effect on hypoxic cells in vivo. It is worthwhile to try this drug in a clinical study

Hypoxic tumor environments exhibit disrupted collagen I fibers and low macromolecular transport.

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Samata M Kakkad

Full Text Available Hypoxic tumor microenvironments result in an aggressive phenotype and resistance to therapy that lead to tumor progression, recurrence, and metastasis. While poor vascularization and the resultant inadequate drug delivery are known to contribute to drug resistance, the effect of hypoxia on molecular transport through the interstitium, and the role of the extracellular matrix (ECM in mediating this transport are unexplored. The dense mesh of fibers present in the ECM can especially influence the movement of macromolecules. Collagen 1 (Col1 fibers form a key component of the ECM in breast cancers. Here we characterized the influence of hypoxia on macromolecular transport in tumors, and the role of Col1 fibers in mediating this transport using an MDA-MB-231 breast cancer xenograft model engineered to express red fluorescent protein under hypoxia. Magnetic resonance imaging of macromolecular transport was combined with second harmonic generation microscopy of Col1 fibers. Hypoxic tumor regions displayed significantly decreased Col1 fiber density and volume, as well as significantly lower macromolecular draining and pooling rates, than normoxic regions. Regions adjacent to severely hypoxic areas revealed higher deposition of Col1 fibers and increased macromolecular transport. These data suggest that Col1 fibers may facilitate macromolecular transport in tumors, and their reduction in hypoxic regions may reduce this transport. Decreased macromolecular transport in hypoxic regions may also contribute to poor drug delivery and tumor recurrence in hypoxic regions. High Col1 fiber density observed around hypoxic regions may facilitate the escape of aggressive cancer cells from hypoxic regions.

Hypoxic preconditioning protects photoreceptors against light damage independently of hypoxia inducible transcription factors in rods.

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Kast, Brigitte; Schori, Christian; Grimm, Christian

2016-05-01

Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lecithin-Bound Iodine Prevents Disruption of Tight Junctions of Retinal Pigment Epithelial Cells under Hypoxic Stress

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Masahiko Sugimoto

2016-01-01

Full Text Available Aim. We investigated whether lecithin-bound iodine (LBI can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia. Method. Cultured human retinal pigment epithelial (ARPE-19 cells were pretreated with LBI. To mimic hypoxic conditions, cells were incubated with CoCl2. We compared the integrity of the tight junctions (TJs of control to cells with either LBI alone, CoCl2 alone, or LBI + CoCl2. The levels of cytokines in the conditioned media were also determined. Results. Significant decrease in the zonula occludens-1 (ZO-1 intensity in the CoCl2 group compared to the control (5787.7 ± 4126.4 in CoCl2 group versus 29244.6 ± 2981.2 in control; average ± standard deviation. But the decrease was not significant in the LBI + CoCl2 (27189.0 ± 11231.1. The levels of monocyte chemoattractant protein-1 (MCP-1 and Chemokine (C-C Motif Ligand 11 (CCL-11 were significantly higher in the CoCl2 than in the control (340.8 ± 43.3 versus 279.7 ± 68.3 pg/mL for MCP-1, and 15.2 ± 12.9 versus 12.5 ± 6.1 pg/mL for CCL-11. With LBI pretreatment, the levels of both cytokines were decreased to 182.6 ± 23.8 (MCP-1 and 5.46 ± 1.9 pg/mL for CCL-11. Blockade of MCP-1 or CCL-11 also shows similar result representing TJ protection from hypoxic stress. Conclusions. LBI results in a protective action from hypoxia.

Impact of perinatal systemic hypoxic-ischemic injury on the brain of male offspring rats: an improved model of neonatal hypoxic-ischemic encephalopathy in early preterm newborns.

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Yuejun Huang

Full Text Available In this study, we attempted to design a model using Sprague-Dawley rats to better reproduce perinatal systemic hypoxic-ischemic encephalopathy (HIE in early preterm newborns. On day 21 of gestation, the uterus of pregnant rats were exposed and the blood supply to the fetuses of neonatal HIE groups were thoroughly abscised by hemostatic clamp for 5, 10 or 15 min. Thereafter, fetuses were moved from the uterus and manually stimulated to initiate breathing in an incubator at 37 °C for 1 hr in air. We showed that survival rates of offspring rats were decreased with longer hypoxic time. TUNEL staining showed that apoptotic cells were significant increased in the brains of offspring rats from the 10 min and 15 min HIE groups as compared to the offspring rats in the control group at postnatal day (PND 1, but there was no statistical difference between the offspring rats in the 5 min HIE and control groups. The perinatal hypoxic treatment resulted in decreased neurons and increased cleaved caspase-3 protein levels in the offspring rats from all HIE groups at PND 1. Platform crossing times and the percentage of the time spent in the target quadrant of Morris Water Maze test were significantly reduced in the offspring rats of all HIE groups at PND 30, which were associated with decreased brain-derived neurotrophic factor levels and neuronal cells in the hippocampus of offspring rats at PND 35. These data demonstrated that perinatal ischemic injury led to the death of neuronal cells and long-lasting impairment of memory. This model reproduced hypoxic ischemic encephalopathy in early preterm newborns and may be appropriate for investigating therapeutic interventions.

Physiologic basis for intermittent hypoxic episodes in preterm infants.

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Martin, R J; Di Fiore, J M; Macfarlane, P M; Wilson, C G

2012-01-01

Intermittent hypoxic episodes are typically a consequence of immature respiratory control and remain a troublesome challenge for the neonatologist. Furthermore, their frequency and magnitude are commonly underestimated by clinically employed pulse oximeter settings. In extremely low birth weight infants the incidence of intermittent hypoxia [IH] progressively increases over the first 4 weeks of postnatal life, with a subsequent plateau followed by a slow decline beginning at weeks six to eight. Over this period of unstable respiratory control, increased oxygen-sensitive peripheral chemoreceptor activity has been associated with a higher incidence of apnea of prematurity. In contrast, infants with bronchopulmonary dysplasia [chronic neonatal lung disease] exhibit decreased peripheral chemosensitivity, although the effect on respiratory stability in this population is unclear. Such episodic hypoxia/reoxygenation in early life has the potential to sustain a proinflammatory cascade with resultant multisystem, including respiratory, morbidity. Therapeutic approaches for intermittent hypoxic episodes comprise careful titration of baseline or supplemental inspired oxygen as well as xanthine therapy to prevent apnea of prematurity. Characterization of the pathophysiologic basis for such intermittent hypoxic episodes and their consequences during early life is necessary to provide an evidence-based approach to their management.

Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions.

Science.gov (United States)

Zhang, Xiaoyu; Li, Shuchen; Li, Mingrong; Huang, Haiying; Li, Jingyuan; Zhou, Changwei

2016-08-01

Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O 2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic

Sirtuin 6 protects the heart from hypoxic damage

International Nuclear Information System (INIS)

Maksin-Matveev, Anna; Kanfi, Yariv; Hochhauser, Edith; Isak, Ahuva; Cohen, Haim Y.; Shainberg, Asher

2015-01-01

Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whether life extension is associated with cardioprotection against hypoxia. The proposed study is to develop approaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate the mechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG) with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyocytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewer damage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactate dehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI) binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechanism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein level of B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level of phospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, which might explain life extension. - Highlights: • Sirtuin 6 is a protein associated with prolonged life expectancy. • Over-expression of sirtuin 6 protects cardiocytes from hypoxia and oxidative stress. • Over-expression of sirtuin 6 activates the pAMPKα pathway and the Bcl2 expression. • Over-expression of sirtuin 6 decreases ROS formation and pAkt level during hypoxia. • These pathways protect cardiocytes from hypoxia and might explain lifespan extension

Sirtuin 6 protects the heart from hypoxic damage

Energy Technology Data Exchange (ETDEWEB)

Maksin-Matveev, Anna; Kanfi, Yariv [The Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900 (Israel); Hochhauser, Edith [The Laboratory of the Department of Cardiothoracic Surgery, Felsenstein Medical Research Center, Rabin Medical Center, Petach Tikva (Israel); Isak, Ahuva; Cohen, Haim Y. [The Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900 (Israel); Shainberg, Asher, E-mail: asher.shainberg@gmail.com [The Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900 (Israel)

2015-01-01

Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whether life extension is associated with cardioprotection against hypoxia. The proposed study is to develop approaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate the mechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG) with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyocytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewer damage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactate dehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI) binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechanism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein level of B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level of phospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, which might explain life extension. - Highlights: • Sirtuin 6 is a protein associated with prolonged life expectancy. • Over-expression of sirtuin 6 protects cardiocytes from hypoxia and oxidative stress. • Over-expression of sirtuin 6 activates the pAMPKα pathway and the Bcl2 expression. • Over-expression of sirtuin 6 decreases ROS formation and pAkt level during hypoxia. • These pathways protect cardiocytes from hypoxia and might explain lifespan extension.

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models.

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Benito, Juliana; Ramirez, Marc S; Millward, Niki Zacharias; Velez, Juliana; Harutyunyan, Karine G; Lu, Hongbo; Shi, Yue-Xi; Matre, Polina; Jacamo, Rodrigo; Ma, Helen; Konoplev, Sergej; McQueen, Teresa; Volgin, Andrei; Protopopova, Marina; Mu, Hong; Lee, Jaehyuk; Bhattacharya, Pratip K; Marszalek, Joseph R; Davis, R Eric; Bankson, James A; Cortes, Jorge E; Hart, Charles P; Andreeff, Michael; Konopleva, Marina

2016-04-01

To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a "hypoxia index" compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. ©2015 American Association for Cancer Research.

Intracapillary HbO2 saturations in murine tumours and human tumour xenografts measured by cryospectrophotometry: relationship to tumour volume, tumour pH and fraction of radiobiologically hypoxic cells.

Science.gov (United States)

Rofstad, E K; Fenton, B M; Sutherland, R M

1988-05-01

Frequency distributions for intracapillary HbO2 saturation were determined for two murine tumour lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) using a cryospectrophotometric method. The aim was to search for possible relationships between HbO2 saturation status and tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Tumour pH was measured by 31P NMR spectroscopy. Hypoxic fractions were determined from cell survival curves for tumours irradiated in vivo and assayed in vitro. Tumours in the volume range 100-4000 mm3 were studied and the majority of the vessels were found to have HbO2 saturations below 10%. The volume-dependence of the HbO2 frequency distributions differed significantly among the four tumour lines; HbO2 saturation status decreased with increasing tumour volume for the KHT, RIF-1 and MLS lines and was independent of tumour volume for the OWI line. The data indicated that the rate of decrease in HbO2 saturation status during tumour growth was related to the rate of development of necrosis. The volume-dependence of tumour pH was very similar to that of the HbO2 saturation status for all tumour lines. Significant correlations were therefore found between HbO2 saturation status and tumour pH, both within tumour lines and across the four tumour lines, reflecting that the volume-dependence of both parameters probably was a compulsory consequence of reduced oxygen supply conditions during tumour growth. Hypoxic fraction increased during tumour growth for the KHT, RIF-1 and MLS lines and was volume-independent for the OWI line, suggesting a relationship between HbO2 saturation status and hypoxic fraction within tumour lines. However, there was no correlation between these two parameters across the four tumour lines, indicating that the hypoxic fraction of a tumour is not determined only by the oxygen supply conditions; other parameters may also be important, e.g. oxygen diffusivity, rate of oxygen

Detection of hypoxic fractions in murine tumors by comet assay: Comparison with other techniques

International Nuclear Information System (INIS)

Hu, Q.; Kavanagh, M.C.; Newcombe, D.

1995-01-01

The alkaline comet assay was used to detect the hypoxic fractions of murine tumors. A total of four tumor types were tested using needle aspiration biopsies taken immediately after a radiation dose of 15 Gy. Initial studies confirmed that the normalized tail moment, a parameter reflecting single-strand DNA breaks induced by the radiation, was linearly related to radiation dose. Further, it was shown that for a mixed population (1:1) of cells irradiated under air-breathing or hypoxic conditions, the histogram of normal tail moment values obtained from analyzing 400 cells in the population had a double peak which, when fitted with two Gaussian distributions, gave a good estimate of the proportion of the two subpopulations. For the four tumor types, the means of the calculated hypoxic fractions from four or five individual tumors were 0.15 ± 0.04 for B16F1, 0.08 ± 0.04 for KHT-LP1, 0.17 ± 0.04 for RIF-1 and 0.04 ± 0.01 for SCCVII. Analysis of variance showed that the hypoxic fraction in KHT-LP1 tumors is significantly lower than those of the other three tumors (P = 0.026) but that there is no significant difference in hypoxic fraction between B16F1, RIF-1 and SCCVII tumors (P = 0.574). Results from multiple samples taken from each of five RIF-1 tumors showed that the intertumor heterogeneity of hypoxic fractions was greater than that within the same tumor. The mean hypoxic fraction obtained using the comet assay for the four tumor types was compared with the hypoxic fraction determined by the clonogenic assay, or median pO 2 values, or [ 3 H]misonidazole binding in the same tumor types. The values of hypoxic fraction obtained with the comet assay were two to four times lower than those measured by the paired survival method. Preliminary results obtained with a dose of 5 Gy were consistent with those obtained using 15 Gy. These results suggest the further development of the comet assay for clinical studies. 21 refs., 7 figs., 5 tabs

Mammalian cell biology

International Nuclear Information System (INIS)

Elkind, M.M.

1975-01-01

Progress is reported on the following research projects: the effects of N-ethyl-maleimide and hydroxyurea on hamster cells in culture; sensitization of synchronized human cells to x rays by N-ethylmaleimide; sensitization of hypoxic mammalian cells with a sulfhydryl inhibitor; damage interaction due to ionizing and nonionizing radiation in mammalian cells; DNA damage relative to radioinduced cell killing; spurious photolability of DNA labeled with methyl- 14 C-thymidine; radioinduced malignant transformation of cultured mouse cells; a comparison of properties of uv and near uv light relative to cell function and DNA damage; Monte Carlo simulation of DNA damage and repair mechanisms; and radiobiology of fast neutrons

Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

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Gao, Yuansheng; Chen, Zhengju; Leung, Susan W S; Vanhoutte, Paul M

2015-06-01

In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

[Effects of intermittent hypoxic exposure on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha and erythropoietin levels].

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Zhang, Cheng-yan; Zhang, Ji-xin; Lü, Xiao-tao; Li, Bao-yu

2009-10-01

To investigate the effects of intermittent hypoxic exposure and normoxic convalescence on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) levels. Rat models of intermittent hypoxic exposure were established, combined with the clinical research on volunteers experiencing the intermittent plateau work. Blood samples for red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (HCT) were collected, serum HIF-1alpha and EPO levels were measured using enzyme linked immunosorbent assay. RBC counts, Hb concentration and HCT were significantly higher than the normoxic group (P hypoxic exposure can enhance serum hypoxia inducible factor-1 alpha and erythropointin levels and the generation of red blood cells, which leads to an increase in hemoglobin concentration and hematocrit. The results have changed with the hypoxic exposure period prolonged. Normoxic convalescence after intermittent hypoxic exposure can make the related indexes reduced, and contribute to the organism recovery.

A novel gene therapy-based approach that selectively targets hypoxic regions within solid tumors

International Nuclear Information System (INIS)

Dougherty, S.T.; Dougherty, G.J.; Davis, P.D.

2003-01-01

There is compelling evidence that malignant cells present within the hypoxic regions that are commonly found within solid tumors contribute significantly to local recurrence following radiation therapy. We describe now a novel strategy designed to target such cells that exploits the differential production within hypoxic regions of the pro-angiogenic cytokine vascular endothelial cell growth factor (VEGF). Specifically, we have generated cDNA constructs that encode two distinct chimeric cell surface proteins that incorporate, respectively, the extracellular domains of the VEGF receptors Flk-1 or Flt-1, fused in frame to the membrane spanning and cytoplasmic domains of the pro-apoptotic protein Fas. Both chimeric proteins (Flk/Fas and Flt/Fas) appear stable and can be readily detected on the surface of transfected cells by Western blot and/or FACS analysis. Importantly, tumor cells expressing the chimeric proteins were rapidly killed in a dose-dependent fashion upon the addition of exogenous recombinant VEGF. Adenoviral vectors encoding Flk/Fas have been generated and shown to induce tumor cells to undergo apoptosis upon transfer to hypoxic conditions in vitro. This activity is dependent upon the endogenous production of VEGF. Studies are currently underway to test the ability of adenoviral Flk/Fas (Ad.Flk/Fas) to reduce tumor recurrence in vivo when used as an adjuvant therapy in conjunction with clinically relevant doses of ionizing radiation

Hyperthermic radiosensitization of hypoxic cells with chlorpromazine

International Nuclear Information System (INIS)

Brown, D.M.; Herman, T.S.; Bump, E.A.; Hahn, G.M.; Calderwood, S.K.

1987-01-01

Many chemical compounds of diverse structures, properties and biological activities have been found to have a profound effect on the survival of mammalian cells in vitro and in more limited studies on murine tumors in vivo when they were administered with hyperthermia (42-44 0 C). These compounds include some of the conventional antitumor chemotherapeutics such as alkylating agents, cisplatinum, adriamycin, bleomcyin and the nitrosoureas. Furthermore, compounds have been identified that are without antitumor activity or cytotoxicity at 37 0 C, but in the presence of elevated temperature, the cytotoxicity is greatly enhanced. These compounds include: aliphatic alcohols; aminothiols such as cysteamine; The polyene antifungal agent Amphotericin B; The local anesthetic procaine; and polycations such as spermine and DEAE-dextran. It is the purpose of this program to extend these initial observations into a dedicated, systematic study that will develop chemical agents in vitro and in vivo to enhance the effectiveness of clinical hyperthermia. Compounds will also be identified that will sensitize tumor cells in vitro and in vivo to suboptimal treatment temperatures (40-42 0 C) and to sensitize thermotolerant cells and tumors to subsequent heat treatments. Data is presented from the authors' initial in vitro evaluation

Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

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Ditte, Peter; Dequiedt, Franck; Svastova, Eliska; Hulikova, Alzbeta; Ohradanova-Repic, Anna; Zatovicova, Miriam; Csaderova, Lucia; Kopacek, Juraj; Supuran, Claudiu T; Pastorekova, Silvia; Pastorek, Jaromir

2011-12-15

In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.

IAEA-HypoX. A randomized multicenter study of the hypoxic radiosensitizer nimorazole concomitant with accelerated radiotherapy in head and neck squamous cell carcinoma

DEFF Research Database (Denmark)

Metwally, Mohamed Ahmed Hassan; Ali, Rubina; Kuddu, Maire

2015-01-01

PURPOSE: To test the hypothesis that radiotherapy (RT) of head and neck squamous cell carcinoma (HNSCC) can be improved by hypoxic modification using nimorazole (NIM) in association with accelerated fractionation. MATERIALS AND METHODS: The protocol was activated in March 2012 as an international...

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

Directory of Open Access Journals (Sweden)

Mayuri Prasad

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

A proteomic view of Caenorhabditis elegans caused by short-term hypoxic stress

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Wu Yonghong

2010-09-01

Full Text Available Abstract Background The nematode Caenorhabditis elegans is both sensitive and tolerant to hypoxic stress, particularly when the evolutionarily conserved hypoxia response pathway HIF-1/EGL-9/VHL is involved. Hypoxia-induced changes in the expression of a number of genes have been analyzed using whole genome microarrays in C. elegans, but the changes at the protein level in response to hypoxic stress still remain unclear. Results Here, we utilized a quantitative proteomic approach to evaluate changes in the expression patterns of proteins during the early response to hypoxia in C. elegans. Two-dimensional difference gel electrophoresis (2D-DIGE was used to compare the proteomic maps of wild type C. elegans strain N2 under a 4-h hypoxia treatment (0.2% oxygen and under normoxia (control. A subsequent analysis by MALDI-TOF-TOF-MS revealed nineteen protein spots that were differentially expressed. Nine of the protein spots were significantly upregulated, and ten were downregulated upon hypoxic stress. Three of the upregulated proteins were involved in cytoskeletal function (LEV-11, MLC-1, ACT-4, while another three upregulated (ATP-2, ATP-5, VHA-8 were ATP synthases functionally related to energy metabolism. Four ribosomal proteins (RPL-7, RPL-8, RPL-21, RPS-8 were downregulated, indicating a decrease in the level of protein translation upon hypoxic stress. The overexpression of tropomyosin (LEV-11 was further validated by Western blot. In addition, the mutant strain of lev-11(x12 also showed a hypoxia-sensitive phenotype in subsequent analyses, confirming the proteomic findings. Conclusions Taken together, our data suggest that altered protein expression, structural protein remodeling, and the reduction of translation might play important roles in the early response to oxygen deprivation in C. elegans, and this information will help broaden our knowledge on the mechanism of hypoxia response.

The Effects of Sympathetic Inhibition on Metabolic and Cardiopulmonary Responses to Exercise in Hypoxic Conditions.

Science.gov (United States)

Scalzo, Rebecca L; Peltonen, Garrett L; Binns, Scott E; Klochak, Anna L; Szallar, Steve E; Wood, Lacey M; Larson, Dennis G; Luckasen, Gary J; Irwin, David; Schroeder, Thies; Hamilton, Karyn L; Bell, Christopher

2015-12-01

Pre-exertion skeletal muscle glycogen content is an important physiological determinant of endurance exercise performance: low glycogen stores contribute to premature fatigue. In low-oxygen environments (hypoxia), the important contribution of carbohydrates to endurance performance is further enhanced as glucose and glycogen dependence is increased; however, the insulin sensitivity of healthy adult humans is decreased. In light of this insulin resistance, maintaining skeletal muscle glycogen in hypoxia becomes difficult, and subsequent endurance performance is impaired. Sympathetic inhibition promotes insulin sensitivity in hypoxia but may impair hypoxic exercise performance, in part due to suppression of cardiac output. Accordingly, we tested the hypothesis that hypoxic exercise performance after intravenous glucose feeding in a low-oxygen environment will be attenuated when feeding occurs during sympathetic inhibition. On 2 separate occasions, while breathing a hypoxic gas mixture, 10 healthy men received 1 hour of parenteral carbohydrate infusion (20% glucose solution in saline; 75 g), after which they performed stationary cycle ergometer exercise (~65% maximal oxygen uptake) until exhaustion. Forty-eight hours before 1 visit, chosen randomly, sympathetic inhibition via transdermal clonidine (0.2 mg/d) was initiated. The mean time to exhaustion after glucose feeding both with and without sympathetic inhibition was not different (22.7 ± 5.4 minutes vs 23.5 ± 5.1 minutes; P = .73). Sympathetic inhibition protects against hypoxia-mediated insulin resistance without influencing subsequent hypoxic endurance performance. Copyright © 2015 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.

A change in the oxygen effect throughout the cell-cycle of human cells of the line NHIK 3025 cultivated in vitro

International Nuclear Information System (INIS)

Pettersen, E.O.; Christensen, T.; Bakke, O.; Oftebro, R.

1977-01-01

NHIK 3025 cells were synchronized by repeated mitotic selection. The S-phase was determined by 3 H-thymidine incorporation and scintillation counting. By comparing the age-response curves of aerobic cells irradiated with 500 rad with those of extremely hypoxic ( 2 ) cells irradiated with 1500 rad, it was found that the sensitizing effect of oxygen was not constant throughout the cycle. It was significantly higher in S, G2 and mitosis than in G1. No significant sensitizing effect of 120 p.p.m. O 2 (compared with 2 ) was found on cells in G1 when the cells were irradiated with 1500 rad. In S, G2 and mitosis, however, the sensitizing effect of oxygen at 120 p.p.m was considered to be significant. Experiment performed with cells irradiated with 2000 rad in contact with either 2 or 80 p.p.m. O 2 showed the same trend, little sensitizing effect in G1 and higher in S, G2 and mitosis. Dose-response curves for cells in mid-G1 and mid-S under aerobic and extremely hypoxic conditions were well fitted by the formula S = exp (-αD-βD 2 ). From the dose-response curves it was concluded that the change in the sensitizing effect of oxygen throughout the cell-cycle only appeared for low doses (in the dose region where α dominates). The sensitizing effect of oxygen on cells in mid-G1 was found to be increasing with increasing dose. (author)

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

International Nuclear Information System (INIS)

Chen, Zhi-Dong; Xu, Liang; Tang, Kan-Kai; Gong, Fang-Xiao; Liu, Jing-Quan; Ni, Yin; Jiang, Ling-Zhi; Hong, Jun; Han, Fang; Li, Qian; Yang, Xiang-Hong; Sun, Ren-Hua; Mo, Shi-Jing

2016-01-01

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβ phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhi-Dong [Department of Critical Care Medicine, The First Affiliated Hospital of Huzhou Normal College, Huzhou 313000, Zhejiang (China); Xu, Liang [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China); Tang, Kan-Kai [Department of Critical Care Medicine, The First Affiliated Hospital of Huzhou Normal College, Huzhou 313000, Zhejiang (China); Gong, Fang-Xiao; Liu, Jing-Quan; Ni, Yin; Jiang, Ling-Zhi; Hong, Jun; Han, Fang; Li, Qian; Yang, Xiang-Hong [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China); Sun, Ren-Hua, E-mail: jqin168@hotmail.com [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China); Mo, Shi-Jing, E-mail: msj860307@163.com [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China)

2016-09-10

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβ phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF

Bumetanide enhances phenobarbital efficacy in a rat model of hypoxic neonatal seizures.

Science.gov (United States)

Cleary, Ryan T; Sun, Hongyu; Huynh, Thanhthao; Manning, Simon M; Li, Yijun; Rotenberg, Alexander; Talos, Delia M; Kahle, Kristopher T; Jackson, Michele; Rakhade, Sanjay N; Berry, Gerard T; Berry, Gerard; Jensen, Frances E

2013-01-01

Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na(+)-K(+)-2 Cl(-) cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABAA receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.

Experimental study on central nervous toxicity of 'misonidazole' a hypoxic cell radiosensitizer

International Nuclear Information System (INIS)

Takahashi, Iku

1981-01-01

'Misonidazole', a radiosensitizer for hypoxic cells is expected to be applied to the treatment of malignant tumors, but its side effect becomes a subject of study, because its effective dose is close to its lethal dose. The auther performed experiments with mice on the central nervous toxicity, which is the most lethal of the side effects of Misonidazole, with the following results; 1. The abrupt death seen after the administration of a large dose of Misonidazole was attributable to the central nervous toxicity. LD 50 for d.d. strain mouse was 1.55 mg per body weight g. 2. The used mice always developed convulsion before death. But the administration of anticonvulsant failed to free them from death. 3. Autopsy findings were such abnormal ones as the degeneration and exfoliation of nerve cells and diapedetic focus. After sacrifice, however, no findings indicative of disturbance of central nerve could be detected. 4. Misonidazole, even in a small divided dose, left intracerebral retention, though slightly, indicating that its accumulation in the brain would be increased with increase in the dose. 5. The disturbance of central nerve was not exacerbated by the whole brain irradiation with Misonidazole. (author)

Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor.

Science.gov (United States)

Kim, So Young; Choi, Yong Jun; Joung, Sun Myung; Lee, Byung Ho; Jung, Yi-Sook; Lee, Joo Young

2010-04-01

Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

Time Domains of the Hypoxic Ventilatory Response and Their Molecular Basis

Science.gov (United States)

Pamenter, Matthew E.; Powell, Frank L.

2016-01-01

Ventilatory responses to hypoxia vary widely depending on the pattern and length of hypoxic exposure. Acute, prolonged, or intermittent hypoxic episodes can increase or decrease breathing for seconds to years, both during the hypoxic stimulus, and also after its removal. These myriad effects are the result of a complicated web of molecular interactions that underlie plasticity in the respiratory control reflex circuits and ultimately control the physiology of breathing in hypoxia. Since the time domains of the physiological hypoxic ventilatory response (HVR) were identified, considerable research effort has gone toward elucidating the underlying molecular mechanisms that mediate these varied responses. This research has begun to describe complicated and plastic interactions in the relay circuits between the peripheral chemoreceptors and the ventilatory control circuits within the central nervous system. Intriguingly, many of these molecular pathways seem to share key components between the different time domains, suggesting that varied physiological HVRs are the result of specific modifications to overlapping pathways. This review highlights what has been discovered regarding the cell and molecular level control of the time domains of the HVR, and highlights key areas where further research is required. Understanding the molecular control of ventilation in hypoxia has important implications for basic physiology and is emerging as an important component of several clinical fields. PMID:27347896

The lethal interaction of x ray and penicillin induced lesions following x-irradiation of Escherichia coli B/r in the presence of hypoxic cell sensitizers

International Nuclear Information System (INIS)

Gillies, N.E.; Obioha, F.I.

1982-01-01

When Escherichia coli B/r were x-irradiated under anoxia in the presence of different electron-affinic sensitizers and then incubated in broth containing penicillin (at a concentration that did not kill unirradiated cells) additional killing of the bacteria occurred provided the sensitizers were of relatively high lipophilicity. The overall effect was to increase the efficiency of these sensitizers. It is concluded that sensitizer-dependent latent radiation lesions(s) are produced in membrane components of the cell envelope that interact with damage caused by penicillin in the peptidoglycan layer and this causes the additional lethality

Correlation of hypoxic cell fraction with glucose metabolic rate in gliomas with 18F-Fluoromisonidazole (FMISO) and 18F- Fluorodeoxyglucose (FDG) positron emission tomography (PET)

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Tauro, A.J.; Scott, A.M.; Hannah, A.; Pathmaraj, K.; Tochon-Danguy, H.; Sachinidis, J.I.; Chan, J.D.; Berlangieri, S.U.; Egan, G.F.; Fabinyi, G.; McKay, W.J.; Cher, L.M.; Austin and Repatriation Medical Centre, Heidelberg, VIC

1998-01-01

Full text: FDG-PET studies of brain tumours to measure tumour activity are well established, with regions of higher grade tumour utilising more glucose compared to lower grade tumour tissue and normal tissue. FDG uptake in tumour cells may reflect anaerobic glycolysis, but this has not been proven in- vivo. FMISO is a novel positron-emitting compound that has been shown to selectively identify hypoxic but viable tissue, which may contribute to chemoradiotherapy resistance in tumour cells. Studies correlating measurements of regional hypoxia and glucose activity within brain tumours prior to therapy may help gain further insight into the relationship between hypoxic tumour tissue and resistance to chemoradiotherapy. Three patients with newly diagnosed primary brain tumours have been prospectively studied with FMISO-PET, FDG-PET and MRI, prior to surgery. Each patient presented with a suspected primary brain glioma on MRI, which were all confirmed to be high grade glioma on subsequent histology at surgery FMISO-PET, FDG-PET and MRI images of all patients were co-registered to precisely identify the areas of metabolic activity within tumour and surrounding cortical tissue. All gliomas demonstrated areas of FMISO uptake, which corresponded to areas of maximal FDG uptake, indicating a correlation between hypoxic areas within tumour with areas of increased glucose metabolic activity. This supports the hypothesis that hypoxic areas within tumour tissue may be associated with increased FDG uptake, although whether hypoxia itself increases FDG uptake remains controversial. These correlative studies characterising areas of hypoxia and glucose activity should hopefully assist in future therapeutic manipulations to improve the outcome from treatment of primary brain tumours

Glutathione requirement for the rejoining of radiation-induced DNA breaks in misonidazole-treated cells

International Nuclear Information System (INIS)

Edgren, M.; Revesz, L.

1985-01-01

The role of glutathione (GSH) in the rejoining of radiation-induced single-strand DNA breaks (ssb) was studied in human fibroblast cultures sensitized to radiation by a 30 min treatment with 1 mM misonidazole (MISO). Hypoxically irradiated cells, deficient in GSH, either inherently, or due to a 16 h incubation with 1 mM buthionine sulphoximine (BSO), rejoined the breaks after MISO treatment at a lower rate and to a lesser extent than did GSH-proficient cells. Without MISO treatment, the hypoxically induced ssb were rejoined in the GSH-deficient cells as effectively as in the proficient cells. It is concluded that a large proportion of the breaks which arise after hypoxic irradiation in the presence of MISO are of a different type to those which arise in the absence of the drug, and require a particular GSH-dependent, enzymatic repair system. This requirement for rejoining in hypoxically irradiated, MISO-treated cells is similar to that seen earlier in MISO-untreated, oxically irradiated cells, and suggests that the ssb induced by radiation in the presence of MISO or oxygen are of a similar nature. (author)

Hypoxic training: Clinical benefits on cardiometabolic risk factors.

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Wee, Justin; Climstein, Mike

2015-01-01

The main aim of this review was to evaluate the effectiveness of hypoxic training on the modulation of cardiometabolic risk factors. Literature review. An electronic search encompassing five databases (PUBMED, EMBASE, MEDLINE, CINAHL, and SPORTDiscus) was conducted. A total of 2138 articles were retrieved. After excluding non-relevant articles, duplications and outcomes not related to cardiometabolic risk factors, 25 articles were chosen for review. Body weight and body composition were reported to be significantly improved when hypoxic training (≥1700 m) was used in conjunction with exercise regimes, at least three times a week, however extreme altitudes (>5000 m) resulted in a loss of fat-free muscle mass. Fasting blood glucose levels generally improved over time (≥21 days) at moderate levels of altitude (1500 m-3000 m), although reductions in blood glucose tolerance were observed when subjects were exposed to extreme hypoxia (>4000 m). Resting systolic and diastolic blood pressure levels improved as much as 26 mmHg and 13 mmHg respectively, with hypoxic training (1285 m-2650 m) in medicated, stable hypertensive subjects. Effects of hypoxic training when used in combination with exercise training on cholesterol levels were mixed. While there were improvements in total cholesterol (-4.2% to -30%) and low-density lipoprotein (-2.6% to -14.3%) reported as a result of hypoxic training, available evidence does not substantiate hypoxic training for the improvement of high-density lipoprotein and triglycerides. In conclusion, hypoxic training may be used as an adjunct treatment to modify some cardiometabolic risk factors. Measurement of hypoxic load may be used to individualize and ascertain appropriate levels of hypoxic training. Copyright © 2013 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Sodium Pyruvate Reduced Hypoxic-Ischemic Injury to Neonatal Rat Brain

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Pan, Rui; Rong, Zhihui; She, Yun; Cao, Yuan; Chang, Li-Wen; Lee, Wei-Hua

2012-01-01

Background Neonatal hypoxia-ischemia (HI) remains a major cause of severe brain damage and is often associated with high mortality and lifelong disability. Immature brains are extremely sensitive to hypoxia-ischemia, shown as prolonged mitochondrial neuronal death. Sodium pyruvate (SP), a substrate of the tricarboxylic acid cycle and an extracellular antioxidant, has been considered as a potential treatment for hypoxic-ischemic encephalopathy (HIE), but its effects have not been evaluated in ...

Hypoxic inactivation of glycogen synthase kinase-3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia-inducible factor-1 signaling.

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Ko, Young San; Cho, Sung Jin; Park, Jinju; Choi, Yiseul; Lee, Jae-Seon; Youn, Hong-Duk; Kim, Woo Ho; Kim, Min A; Park, Jong-Wan; Lee, Byung Lan

2016-09-01

Since the molecular mechanism of hypoxic adaptation in cancer cells is cell-type specific, we investigated whether glycogen synthase kinase-3β (GSK-3β) activation is involved in hypoxia-induced gastric tumor promotion. Stable gastric cancer cell lines (SNU-638, SNU-484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild-type GSK-3β (WT-GSK-3β) or kinase-dead mutant of GSK-3β (KD-GSK-3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK-3β activation in gastric cancer cells. Cell viability and the expressions of HIF-1α protein and VEGF mRNA in gastric cancer cells were higher in KD-GSK-3β transfectants than in WT-GSK-3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF-1α activation, and VEGF expression were higher in KD-GSK-3β tumors than in WT-GSK-3β tumors in vivo. In addition, the expression of hypoxia-induced HIF-1α protein was regulated by GSK-3β at the translational level. Our data suggest that GSK-3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF-1α/VEGF signaling pathway. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Hypoxic preconditioning with cobalt of bone marrow mesenchymal stem cells improves cell migration and enhances therapy for treatment of ischemic acute kidney injury.

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Xiaofang Yu

Full Text Available Mesenchymal stem cell (MSC administration is known to enhance the recovery of the kidney following injury. Here we tested the potential of hypoxic-preconditioned-MSC transplantation to enhance the efficacy of cell therapy on acute kidney injury (AKI by improving MSC migration to the injured kidney. Cobalt was used as hypoxia mimetic preconditioning (HMP. MSC were subjected to HMP through 24 h culture in 200 µmol/L cobalt. Compared to normoxia cultured MSC (NP-MSC, HMP significantly increased the expression of HIF-1α and CXCR4 in MSC and enhanced the migration of MSC in vitro. This effect was lost when MSC were treated with siRNA targeting HIF-1α or CXCR4 antagonist. SPIO labeled MSC were administered to rats with I/R injury followed immediately by magnetic resonance imaging. Imaging clearly showed that HMP-MSC exhibited greater migration and a longer retention time in the ischemic kidney than NP-MSC. Histological evaluation showed more HMP-MSC in the glomerular capillaries of ischemic kidneys than in the kidneys receiving NP-MSC. Occasional tubules showed iron labeling in the HMP group, while no tubules had iron labeling in NP group, indicating the possibility of tubular transdifferentiation after HMP. These results were also confirmed by fluorescence microscopy study using CM-DiI labeling. The increased recruitment of HMP-MSC was associated with reduced kidney injury and enhanced functional recovery. This effect was also related to the increased paracrine action by HMP-MSC. Thus we suggest that by enhancing MSC migration and prolonging kidney retention, hypoxic preconditioning of MSC may be a useful approach for developing AKI cell therapy.

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

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Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

2012-08-01

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Induction of long noncoding RNA MALAT1 in hypoxic mice

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Lelli A

2015-10-01

Full Text Available Aurelia Lelli,1,2,* Karen A Nolan,1,2,* Sara Santambrogio,1,2 Ana Filipa Gonçalves,1,2 Miriam J Schönenberger,1,2 Anna Guinot,1,2 Ian J Frew,1,2 Hugo H Marti,3 David Hoogewijs,1,2,4 Roland H Wenger1,2 1Institute of Physiology and Zurich Center for Human Physiology (ZIHP, University of Zurich, Zurich, Switzerland; 2National Center of Competence in Research "Kidney.CH", Zurich, Switzerland; 3Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany; 4Institute of Physiology, University of Duisburg-Essen, Essen, Germany *These authors contributed equally to this work Abstract: Long thought to be “junk DNA”, in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA. Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1 appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary

In vivo evidence suggesting reciprocal renal hypoxia-inducible factor-1 upregulation and signal transducer and activator of transcription 3 activation in response to hypoxic and non-hypoxic stimuli.

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Nechemia-Arbely, Yael; Khamaisi, Mogher; Rosenberger, Christian; Koesters, Robert; Shina, Ahuva; Geva, Carmit; Shriki, Anat; Klaus, Stephen; Rosen, Seymour; Rose-John, Stefan; Galun, Eithan; Axelrod, Jonathan H; Heyman, Samuel N

2013-04-01

In vitro studies suggest that combined activation of hypoxia-inducible factor (HIF) and signal transducer and activator of transcription 3 (STAT3) promotes the hypoxia response. However, their interrelationship in vivo remains poorly defined. The present study investigated the possible relationship between HIF-1 upregulation and STAT3 activation in the rodent kidney in vivo. Activation of HIF-1 and STAT3 was analysed by immunohistochemical staining and western blot analysis in: (i) models of hypoxia-associated kidney injury induced by radiocontrast media or rhabdomyolysis; (ii) following activation of STAT3 by the interleukin (IL)-6-soluble IL-6 receptor complex; or (iii) following HIF-1α stabilization using hypoxic and non-hypoxic stimuli (mimosine, FG-4497, CO, CoCl(2)) and in targeted von Hippel-Lindau-knockout mice. Western blot analysis and immunostaining revealed marked induction of both transcription factors under all conditions tested, suggesting that in vivo STAT3 can trigger HIF and vice versa. Colocalization of HIF-1α and phosphorylated STAT3 was detected in some, but not all, renal cell types, suggesting that in some cells a paracrine mechanism may be responsible for the reciprocal activation of the two transcription factors. Nevertheless, in several cell types spatial concordance was observed under the majority of conditions tested, suggesting that HIF-1 and STAT3 may act as cotranscription factors. These in vivo studies suggest that, in response to renal hypoxic-stress, upregulation of HIF-1 and activation of STAT3 may be both reciprocal and cell type dependent. © 2013 The Authors Clinical and Experimental Pharmacology and Physiology © 2013 Wiley Publishing Asia Pty Ltd.

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion

International Nuclear Information System (INIS)

Schans, G.P. van der; Vos, O.; Roos-Verheij, W.S.D.; Lohman, P.H.M.

1986-05-01

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and NPSH (non-protein-bound SH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand DNA breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20% for the 3 types of radiation damage. Also under severely hypoxic conditions (0.01 μM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30%). At somewhat higher concentrations of oxygen (0.5-10 μM) however, the sensitization amounted to about 90% for the induction of ssb and dsb and about 50% for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen. (Auth.)

Bumetanide enhances phenobarbital efficacy in a rat model of hypoxic neonatal seizures.

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Ryan T Cleary

Full Text Available Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na(+-K(+-2 Cl(- cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABAA receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.

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

International Nuclear Information System (INIS)

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

2003-01-01

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

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

Science.gov (United States)

Pissarek, M; Reichelt, C; Krauss, G J; Illes, P

1998-11-23

/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 microM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of KATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival viaprotein and DNA synthesis.

Transformed human mesenchymal stem cells are more radiosensitive compared to their cells of origin in normoxia and in physiological hypoxia

International Nuclear Information System (INIS)

Worku, M.; Fersht, N.; Martindale, C.; Funes, J.M.; Shah, S.; Short, S.C.

2013-01-01

The full text of the publication follows. Purpose: The presence of hypoxic regions in tumours is associated with the recurrence of solid tumours after treatment with radiotherapy and thought to be an important element in defining the stem cell niche. We studied the effect of hypoxia on the response to radiation in sequentially transformed human mesenchymal stem cells (MSC) to investigate how the genetic events that lead to tumorigenicity influence the cellular response to radiation under hypoxic and normoxic conditions. Experimental Design: Human bone marrow derived SH2+, SH4+, Stro-1+ MSC were transformed step-wise by retroviral transfection of hTERT, HPV-16 E6 and E7, SV40 small T antigen and oncogenic H-ras. Cells were grown and irradiated with 0, 1 to 5 Gy, X-Ray at 20%, 5% and 1% oxygen tensions. Cytotoxicity, DNA double-strand break (DSB) repair and checkpoint signalling were compared between cells at three different stages of transformation and in different oxygen concentrations. Results: MSCs became more radiosensitive at each point during step-wise transformation, and this effect persisted when cells were irradiated in physiological hypoxia. Increased cytotoxicity of radiation was associated with increased residual DNA DSB at 24 post X-irradiation assessed by gamma-H2AX foci. Growth and irradiation in 1% but not 5% oxygen promoted increased radioresistance compared to growth in 20% oxygen but did not change the relative sensitivity of tumorigenic cells compared to parental cells. Activation of checkpoint signalling before and after single radiation doses is more marked in tumorigenic cells compared to parental lines, and is not altered when cells are irradiated and grown in hypoxic conditions. Conclusions: These data show that tumorigenic cells are more radiosensitive compared to non-tumorigenic parental cells in both normoxic and hypoxic conditions. 1% hypoxia promotes radioresistance in all cells. Checkpoint signalling is up-regulated in tumorigenic

[Autonomic regulation at emotional stress under hypoxic conditions in the elderly with physiological and accelerated aging: effect of hypoxic training].

Science.gov (United States)

Os'mak, E D; Asanov, É O

2014-01-01

The effect of hypoxic training on autonomic regulation in psycho-emotional stress conditions in hypoxic conditions in older people with physiological (25 people) and accelerated (28 people) aging respiratory system. It is shown that hypoxic training leads to an increase in vagal activity indicators (HF) and reduced simpatovagal index (LF/HF), have a normalizing effect on the autonomic balance during stress loads in older people with different types of aging respiratory system.

Temporal Transcriptome of Mouse ATDC5 Chondroprogenitors Differentiating under Hypoxic Conditions

DEFF Research Database (Denmark)

Chen, Li; Fink, Trine; Ebbesen, Peter

2006-01-01

The formation of cartilage takes place in vivo in an environment of reduced oxygen tension. To study the effect of hypoxia on the process of chondrogenesis, ATDC5 mouse chondroprogenitor cells were induced to differentiate by the addition of insulin and cultured under ambient and hypoxic conditions...

Astrocyte-derived proinflammatory cytokines induce hypomyelination in the periventricular white matter in the hypoxic neonatal brain.

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Yiyu Deng

Full Text Available Hypoxic exposure in the perinatal period causes periventricular white matter damage (PWMD, a condition associated with myelination abnormalities. Under hypoxic conditions, glial cells were activated and released a large number of inflammatory mediators in the PWM in neonatal brain, which may result in oligodendrocyte (OL loss and axonal injury. This study aims to determine if astrocytes are activated and generate proinflammatory cytokines that may be coupled with the oligodendroglial loss and hypomyelination observed in hypoxic PWMD. Twenty-four 1-day-old Wistar rats were exposed to hypoxia for 2 h. The rats were then allowed to recover under normoxic conditions for 7 or 28 days before being killed. Another group of 24 rats kept outside the chamber was used as age-matched controls. Upregulated expression of TNF-α and IL-1β was observed in astrocytes in the PWM of P7 hypoxic rats by double immunofluorescence, western blotting and real time RT-PCR. This was linked to apoptosis and enhanced expression of TNF-R1 and IL-1R1 in APC(+ OLs. PLP expression was decreased significantly in the PWM of P28d hypoxic rats. The proportion of myelinated axons was markedly reduced by electron microscopy (EM and the average g-ratios were higher in P28d hypoxic rats. Upregulated expression of TNF-α and IL-1β in primary cultured astrocytes as well as their corresponding receptors in primary culture APC(+ oligodendrocytes were detected under hypoxic conditions. Our results suggest that following a hypoxic insult, astrocytes in the PWM of neonatal rats produce inflammatory cytokines such as TNF-α and IL-1β, which induce apoptosis of OLs via their corresponding receptors associated with them. This results in hypomyelination in the PWM of hypoxic rats.

Elevation of circulating miR-210-3p in high-altitude hypoxic environment

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Yan eYan

2016-03-01

Full Text Available Background: The induction of miR-210-3p, a master hypoxamir, is a consistent feature of the hypoxic response in both normal and malignant cells. However, whether miR-210-3p acts as a circulating factor in response to a hypoxic environment remains unknown. The current study aimed to examine the effect of a high-altitude hypoxic environment on circulating miR-210-3p.Methods: We examined and compared the levels of miR-210-3p using TaqMan-based qRT-PCR in both peripheral blood cells and plasma from 84 ethnic Chinese Tibetans residing at 3560 m, 46 newly arrived migrant Han Chinese (Tibet Han and 82 Han Chinese residing at 8.9 m (Nanjing Han. Furthermore, we analyzed the correlations of miR-210-3p with hematological indices. Results: The relative concentrations of miR-210-3p to internal reference U6 in blood cells were significantly higher in the Tibet Han group (1.01±0.11, P<0.001 and in the Tibetan group (1.17±0.09, P<0.001 than in the Nanjing Han group (0.51±0.04. The absolute concentrations of plasma miR-210-3p were also markedly elevated in the Tibet Han group (503.54±42.95 fmol/L, P=0.004 and in the Tibetan group (557.78±39.84 fmol/L, P<0.001 compared to the Nanjing Han group (358.39±16.16 fmol/L. However, in both blood cells and plasma, miR-210-3p levels were not significantly different between the Tibet Han group and the Tibetan group (P=0.280, P=0.620, respectively. Plasma miR-210-3p concentrations were positively correlated with miR-210-3p levels in blood cells (r=0.192, P=0.005. Furthermore, miR-210-3p levels in both blood cells and plasma showed strong positive correlations with red blood cell counts and hemoglobin and hematocrit values. Conclusion: These data demonstrated, for the first time, that miR-210-3p might act as a circulating factor in response to hypoxic environments and could be associated with human adaptation to life at high altitudes.

Enhancement of the radiation-lethal effect of hypoxic cancer cells by some nitroheterocyclic compounds. Part of a coordinated programme on the improvement of radiotherapy of cancer using modifiers of radiosensitivity of cells

International Nuclear Information System (INIS)

Chiricuta, I.

1981-12-01

The possibilities to enhance the lethal effect of ionizing radiation on hypoxic cells by electron-affinic compounds have stimulated the investigations for finding new chemicals with radiobiological and pharmacological features as adequate as possible. On the other hand, the experimental studies and clinical trials had shown that the aerobic toxicity seems to be the major limiting factor in the use of large doses of radiosensitizers required to achieve significant therapeutic efficiency. The investigations in the present paper were attempted to join these two main directions of research and comprised the syntheses of new nitroheterocyclic compounds with potential radiosensitization properties and the knowledge of biochemical alterations involved in the producing of aerobic toxicity of radiosensitizers aiming to find practical solutions to enhance the efficiency of radiotherapy. Several newly synthesized compounds were tested for their radiosensitizing effect. The experiments carried out on hypoxic cells V 79 showed that only 1-(hydroxyethyl-2'-phosphate)-2-methyl-5-nitroimidazole, dipotassium salt displayed an enhancement ratio of 1.17 (at 8 mM), but lower than in case of parent compound, metronidazole (enhancement ratio = 1.53). It was shown that hypoxic cell radiosensitizers interfere with the cellular energy metabolism. These interferences were found dependent on the electron affinity of drugs. In addition, those radiosensitizers producing a decrease in oxygen consumption caused a supplementary oxygenation of both normal and tumour tissues. It is concluded that the improvement of therapeutic efficiency of radiosensitizers by reducing their aerobic toxicity might be achieved by diminishing their effects on the energy metabolism or by the stimulation of this metabolism and restoration of tissue redox equilibrium

Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

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Sho Kitamoto

Full Text Available MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs; however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α-dependent pathway in some pancreatic cancer cells (e.g., AsPC1, whereas other pancreatic cancer cells (e.g., BxPC3 exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3', a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

Cellular radiation effects and hyperthermia cell cycle kinetics of radiation sensitive mutants of saccharomyces cerevisiae after x-irradiation and hyperthermia

International Nuclear Information System (INIS)

Fingerhut, R.; Kiefer, J.; Otto, F.

1983-01-01

Radiosensitive mutants rad2, rad9, and rad51 of Saccharomyces cerevisiae were X-irradiated with 120 Gy or 60 Gy, heated at 50 0 C for 30 min or treated with a combination of both and incubated in nutrient medium at 30 0 C. Cell number, percentage of budding cells, and cell cycle progression were determined in 45-min intervals. Cell cycle kinetics were investigated by flow cytofluorometry. Hyperthermia leads mainly to a lengthening of G1, whereas X-rays arrest cells of the rad2 and rad9 mutant in G2 and the rad51 - mutant additionaly in a state with DNA contents above G2. Cell division dealy is influenced by oxygen in all strains but to a lesser extent in the rad2 mutant. The effect of the combined treatment appears to be merely additive in the rad2 and rad9 mutant while the rad51 mutant is sensitized to X-irradiation by hyperthermia. No selective action of hyperthermia on hypoxic cells was found. (orig.)

Radiosensitivity and effect of hypoxia in HPV positive head and neck cancer cells

International Nuclear Information System (INIS)

Sørensen, Brita Singers; Busk, Morten; Olthof, Nadine; Speel, Ernst-Jan; Horsman, Michael R.; Alsner, Jan; Overgaard, Jens

2013-01-01

Background and purpose: HPV associated Head and Neck Squamous Cell Carcinoma (HNSCC) represents a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. Previous data from the randomized DAHANCA 5 trial indicated that HPV positive tumors did not benefit from hypoxic modifications by Nimorazole during radiotherapy, whereas a significant benefit was observed in the HPV negative tumors. However, more studies have demonstrated equal frequencies of hypoxic tumors among HPV-positive and HPV-negative tumors. The aim of the present study was to determine radiosensitivity, the impact of hypoxia and the effect of Nimorazole in HPV positive and HPV negative cell lines. Materials and method: The used cell lines were: UDSCC2, UMSCC47 and UPCISCC90 (HPV positive) and FaDu DD , UTSCC33 and UTSCC5 (HPV negative). Cells were cultured under normoxic or hypoxic conditions, and gene expression levels of previously established hypoxia induced genes were assessed by qPCR. Cells were irradiated with various doses under normoxia, hypoxia or hypoxia +1 mM Nimorazole, and the clonogenic survival was determined. Results: The HPV positive and HPV negative cell lines exhibited similar patterns of upregulation of hypoxia induced genes in response to hypoxia. The HPV positive cell lines were up to 2.4 times more radiation sensitive than HPV negative cell lines. However, all HPV positive cells displayed the same response to hypoxia in radiosensitivity, with an OER in the range 2.3–2.9, and a sensitizer effect of Nimorazole of 1.13–1.29, similar to HPV negative cells. Conclusions: Although HPV positive cells had a markedly higher radiosensitivity compared to HPV negative cells, they displayed the same relative radioresistance under hypoxia and the same relative sensitizer effect of Nimorazole. The clinical observation that HPV positive patients do not seem to benefit from Nimorazole treatment is not due to inherent differences in hypoxia sensitivity

Interleukin-17 limits hypoxia-inducible factor 1α and development of hypoxic granulomas during tuberculosis.

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Domingo-Gonzalez, Racquel; Das, Shibali; Griffiths, Kristin L; Ahmed, Mushtaq; Bambouskova, Monika; Gopal, Radha; Gondi, Suhas; Muñoz-Torrico, Marcela; Salazar-Lezama, Miguel A; Cruz-Lagunas, Alfredo; Jiménez-Álvarez, Luis; Ramirez-Martinez, Gustavo; Espinosa-Soto, Ramón; Sultana, Tamanna; Lyons-Weiler, James; Reinhart, Todd A; Arcos, Jesus; de la Luz Garcia-Hernandez, Maria; Mastrangelo, Michael A; Al-Hammadi, Noor; Townsend, Reid; Balada-Llasat, Joan-Miquel; Torrelles, Jordi B; Kaplan, Gilla; Horne, William; Kolls, Jay K; Artyomov, Maxim N; Rangel-Moreno, Javier; Zúñiga, Joaquín; Khader, Shabaana A

2017-10-05

Mycobacterium tuberculosis (Mtb) is a global health threat, compounded by the emergence of drug-resistant strains. A hallmark of pulmonary tuberculosis (TB) is the formation of hypoxic necrotic granulomas, which upon disintegration, release infectious Mtb. Furthermore, hypoxic necrotic granulomas are associated with increased disease severity and provide a niche for drug-resistant Mtb. However, the host immune responses that promote the development of hypoxic TB granulomas are not well described. Using a necrotic Mtb mouse model, we show that loss of Mtb virulence factors, such as phenolic glycolipids, decreases the production of the proinflammatory cytokine IL-17 (also referred to as IL-17A). IL-17 production negatively regulates the development of hypoxic TB granulomas by limiting the expression of the transcription factor hypoxia-inducible factor 1α (HIF1α). In human TB patients, HIF1α mRNA expression is increased. Through genotyping and association analyses in human samples, we identified a link between the single nucleotide polymorphism rs2275913 in the IL-17 promoter (-197G/G), which is associated with decreased IL-17 production upon stimulation with Mtb cell wall. Together, our data highlight a potentially novel role for IL-17 in limiting the development of hypoxic necrotic granulomas and reducing disease severity in TB.

Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

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Cammarata, Patrick R; Neelam, Sudha; Brooks, Morgan M

2015-01-01

The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. Recently, we reported

Reduction of nitric oxide level enhances the radiosensitivity of hypoxic non-small cell lung cancer

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Saleem, Wael; Suzuki, Yoshiyuki; Mobaraki, Abdulelah; Yoshida, Yukari; Noda, Shinei; Saitoh, Jun-ichi; Nakano, Takashi

2011-01-01

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (E-TKI) resistance has emerged as an important clinical issue. To overcome this resistance, researchers have examined different modalities, either for use as a monotherapy or in combination with E-TKI therapy. In the present study, we investigated whether a decrease in nitric oxide (NO) levels affects the radiosensitization of non-small cell lung cancer (NSCLC) cell lines. A549 and H3255 NSCLC cells were examined. They were subjected to hypoxic conditions and monotherapy, or combined therapy using radiation and N G -monomethyl- L -arginine, monoacetate (LNMMA). Reductions in nitric oxide levels enhanced the radiosensitivity of both cell lines and significantly reduced the expression of both hypoxia-inducible factor-1α (HIF-1α) and EGFR in H3255 cells compared to A549 cells. Since NO is significantly associated with cell metabolism, we measured the levels of pyruvate dehydrogenase kinase-1 (PDK-1), reactive oxygen species, and oxygen and observed that the expression of PDK-1 was significantly reduced. This reduction was seen simultaneously after the silencing of HIF-1α; however, not following LNMMA treatment. The oxygen concentration was significantly increased in the treated cells, and their viability decreased in parallel. Reactive oxygen species were decreased after LNMMA and radiation treatment. Adding EGFR-TKI to cells with reduced NO levels further suppressed cell viability when combined with radiation. This study suggests that a reduction in the NO level might substantially overcome the radioresistance of mutant NSCLC cells. (author)

The toxicokinetics of cadmium in carp under normoxic and hypoxic conditions

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Hattink, Jasper; Boeck, Gudrun De; Blust, Ronny

2005-01-01

Temporal depletion of oxygen, i.e. hypoxic events, frequently occurs in natural waters. It has been suggested that accumulation of micro-pollutants increases in aquatic animals as a result of an increased ventilation rate during such occasions. The observed increased toxicity under hypoxia of micro-pollutants may support this hypothesis, but for heavy metals the available uptake studies are contradictory. The present study tests whether accumulation of cadmium in common carp, Cyprinus carpio (L.) is increased under hypoxia and if the toxicokinetics are altered. A cadmium toxicity study was performed in which the cadmium uptake rates were determined using the radiotracer 109 Cd under hypoxia and normoxia. The cadmium toxicokinetics were studied with radiotracer experiments at 100% air saturation, 50%, and 25% saturation from 6.5 nmol/L Cd at 25 deg C. We could confirm the higher sensitivity of carp to cadmium under hypoxia. Hypoxic conditions did not influence the uptake rates or the accumulation dynamics. Therefore, the increased sensitivity of carp for Cd under hypoxia cannot be explained by a higher Cd body burden, initiated by a higher uptake rate or lower elimination rate under hypoxia. Additional, possible indirect effects, such as internal anoxia due to gill damage, could play a role in Cd toxicity under hypoxia

The toxicokinetics of cadmium in carp under normoxic and hypoxic conditions

Energy Technology Data Exchange (ETDEWEB)

Hattink, Jasper [University of Antwerp, Department of Biology, Laboratory of Ecophysiology, Biochemistry, and Toxicology, Groenenborgerlaan 171, 2020 Antwerp (Belgium)]. E-mail: jasper.hattink@ua.ac.be; Boeck, Gudrun De [University of Antwerp, Department of Biology, Laboratory of Ecophysiology, Biochemistry, and Toxicology, Groenenborgerlaan 171, 2020 Antwerp (Belgium); Blust, Ronny [University of Antwerp, Department of Biology, Laboratory of Ecophysiology, Biochemistry, and Toxicology, Groenenborgerlaan 171, 2020 Antwerp (Belgium)

2005-10-05

Temporal depletion of oxygen, i.e. hypoxic events, frequently occurs in natural waters. It has been suggested that accumulation of micro-pollutants increases in aquatic animals as a result of an increased ventilation rate during such occasions. The observed increased toxicity under hypoxia of micro-pollutants may support this hypothesis, but for heavy metals the available uptake studies are contradictory. The present study tests whether accumulation of cadmium in common carp, Cyprinus carpio (L.) is increased under hypoxia and if the toxicokinetics are altered. A cadmium toxicity study was performed in which the cadmium uptake rates were determined using the radiotracer {sup 109}Cd under hypoxia and normoxia. The cadmium toxicokinetics were studied with radiotracer experiments at 100% air saturation, 50%, and 25% saturation from 6.5 nmol/L Cd at 25 deg C. We could confirm the higher sensitivity of carp to cadmium under hypoxia. Hypoxic conditions did not influence the uptake rates or the accumulation dynamics. Therefore, the increased sensitivity of carp for Cd under hypoxia cannot be explained by a higher Cd body burden, initiated by a higher uptake rate or lower elimination rate under hypoxia. Additional, possible indirect effects, such as internal anoxia due to gill damage, could play a role in Cd toxicity under hypoxia.

A novel class of antitumor prodrug, 1-(2'-oxopropyl)-5-fluorouracil (OFU001), that releases 5-fluorouracil upon hypoxic irradiation

International Nuclear Information System (INIS)

Shibamoto, Yuta; Zhou, Ling; Hatta, Hiroshi; Mori, Mayuko; Nishimoto, Sei-ichi

2000-01-01

We have been developing prodrugs of anticancer agents such as 5-fluorouracil (5-FU) that are activated by irradiation under hypoxic conditions via one-electron reduction. Among them, OFU001 [1-(2'-oxopropyl)-5-fluorouracil] is a prototype radiation-activated prodrug. In this study, we investigated the radiation chemical reactivity and the biological effects of OFU001. This prodrug is presumed to release 5-FU through incorporation of hydrated electrons into the antibonding σ * orbital of the C(1')-N(1) bond. Hydrated electrons are active species derived from radiolysis of water, but are readily deactivated by O 2 into superoxide anion radicals (O 2 - ·) under conditions of aerobic irradiation. Therefore, 5-FU release occurs highly specifically upon irradiation under hypoxic conditions. OFU001 dissolved in phosphate buffer released 5-FU with a G-value (mol number of molecules that are decomposed or produced by 1 J of absorbed radiation energy) of 1.9 x 10 -7 mol/J following hypoxic irradiation, while the G-value for 5-FU release was 1.0 x 10 -8 mol/J following aerobic irradiation. However, the G-values for decomposition of OFU001 were almost the same, i.e., 3.4 x 10 -7 mol/J following hypoxic irradiation and 2.5 x 10 -7 mol/J following aerobic irradiation. When hypoxically irradiated (7.5-30 Gy) OFU001 was added to murine SCCVII cells for 1-24 h, a significant cell-killing effect was observed. The degree of this cytotoxicity was consistent with that of authentic 5-FU at the corresponding concentrations. On the other hand, cytotoxicity was minimal when the cells were treated with aerobically irradiated or unirradiated OFU001. This compound had no radiosensitizing effect against SCCVII cells under either aerobic or hypoxic conditions when the drug was removed immediately after irradiation. Since hypoxia is generally most marked in tumors and irradiation is applied at the tumor site, this concept of prodrug design appears to be potentially useful for selective tumor

Nicotinamide and other benzamide analogs as agents for overcoming hypoxic cell radiation resistance in tumours

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Horsman, M.

1996-01-01

Oxygen deficient hypoxic cells, which are resistant to sparsely ionising radiation, have now been identified in most animal and some human solid tumours and will influence the response of those tumours to radiation treatment. This hypoxia can be either chronic, arising from an oxygen diffusion limitation, or acute, resulting from transient stoppages in microregional blood flow. Extensive experimental studies, especially in the last decade, have shown that nicotinamide and structurally related analogs can effectively sensitize murine tumours to both single and fractionated radiation treatments and that they do so in preference to the effects seen in mouse normal tissues. The earliest studies suggested that this enhancement of radiation damage was the result of an inhibition of the repair mechanisms. However, recent studies in mouse tumours have shown that these drugs prevent transient cessations in blood flow, thus inhibiting the development of acute hypoxia. This novel discovery led to the suggestion that the potential role of these agents as radiosensitizers would be when combined with treatments that overcame chronic hypoxia. The combined nicotinamide with hyperthermia proved that the enhancement of radiation damage by both agents together was greater than that seen with each agent alone. Similar results were later seen for nicotinamide combined with a perfluorochemical emulsion, carbogen breathing, and pentoxifylline, and in all these studies the effects in tumours were always greater than those seen in appropriate normal tissues. Of all the analogs, it is nicotinamide itself which has been the most extensively studied as a radiosensitizer in vivo and the one that shows the greatest effect in animal tumours. It is also an agent that has been well established clinically, with daily doses of up to 6 g, associated with a low incidence of side effects. This human dose is equivalent to 100-200 mg/kg in mice and such doses will maximally sensitize murine tumours to

Post-hypoxic recovery of respiratory rhythm generation is gender dependent.

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Alfredo J Garcia

Full Text Available The preBötzinger complex (preBötC is a critical neuronal network for the generation of breathing. Lesioning the preBötC abolishes respiration, while when isolated in vitro, the preBötC continues to generate respiratory rhythmic activity. Although several factors influence rhythmogenesis from this network, little is known about how gender may affect preBötC function. This study examines the influence of gender on respiratory activity and in vitro rhythmogenesis from the preBötC. Recordings of respiratory activity from neonatal mice (P10-13 show that sustained post-hypoxic depression occurs with greater frequency in males compared to females. Moreover, extracellular population recordings from the preBötC in neonatal brainstem slices (P10-13 reveal that the time to the first inspiratory burst following reoxygenation (TTFB is significantly delayed in male rhythmogenesis when compared to the female rhythms. Altering activity of ATP sensitive potassium channels (KATP with either the agonist, diazoxide, or the antagonist, tolbutamide, eliminates differences in TTFB. By contrast, glucose supplementation improves post-hypoxic recovery of female but not male rhythmogenesis. We conclude that post-hypoxic recovery of respiration is gender dependent, which is, in part, centrally manifested at the level of the preBötC. Moreover, these findings provide potential insight into the basis of increased male vulnerability in a variety of conditions such as Sudden Infant Death Syndrome (SIDS.

Dye Sensitized Solar Cell, DSSC

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Pongsatorn Amornpitoksuk

2003-07-01

Full Text Available A dye sensitized solar cell is a new type of solar cell. The operating system of this solar cell type is similar to plant’s photosynthesis process. The sensitizer is available for absorption light and transfer electrons to nanocrystalline metal oxide semiconductor. The ruthenium(II complexes with polypyridyl ligands are usually used as the sensitizers in solar cell. At the present time, the complex of [Ru(2,2',2'’-(COOH3- terpy(NCS3] is the most efficient sensitizer. The total photon to current conversion efficiency was approximately 10% at AM = 1.5.

A behavioural study of neuroglobin-overexpressing mice under normoxic and hypoxic conditions

NARCIS (Netherlands)

Van Leuven, Wendy; Van Dam, Debby; Moens, Luc; De Deyn, Peter Paul; Dewilde, Sylvia

Neuroglobin (Ngb), a neuron-specific heme-binding protein that binds O-2, CO and NO reversibly, and promotes in vivo and in vitro cell survival after hypoxic and ischaemic insult Although the mechanisms of this neuroprotection remain unknown, Ngb might play an important role in counteracting the

X irradiation combined with TNF alpha-related apoptosis-inducing ligand (TRAIL) reduces hypoxic regions of human gastric adenocarcinoma xenografts in SCID mice

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Takahashi, Momoko; Yasui, Hironobu; Ogura, Aki; Asanuma, Taketoshi; Inanami, Osamu; Kubota, Nobuo; Tsujitani, Michihiko; Kuwabara, Mikinori

2008-01-01

Our previous study showed that X irradiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines under not only normoxia but also hypoxia. X irradiation combined with TNF α-related apoptosis-inducing ligand (TRAIL), which is the ligand of DR5, induced apoptosis in vitro (Takahashi et al., (2007) Journal of Radiation Research, 48: 461-468). In this report, we examined the in vivo antitumor efficacy of X irradiation combined with TRAIL treatment in tumor xenograft models derived from human gastric adenocarcinoma MKN45 and MKN28 cells in severe combined immunodeficiency (SCID) mice. X irradiation combined with TRAIL synergistically suppressed the tumor growth rates in the xenograft models derived from MKN45 and MKN28 cells, which have wild type Tp53 and mutated Tp53, respectively, indicating that the antitumor effects occurred in a Tp53-independent manner. Histological analysis showed that the combination of X irradiation and TRAIL induced caspase-3-dependent apoptotic cell death. Moreover, the immunohistochemical detection of hypoxic regions using the hypoxic marker pimonidazole revealed that caspase-3-dependent apoptosis occurred in the hypoxic regions in the tumors. These results indicated that X irradiation combined with TRAIL may be a useful treatment to reduce tumor growth in not only normoxic but also hypoxic regions. (author)

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

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Germain, Todd; Ansari, Megan; Pappas, Dimitri

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

CT diagnosis of hypoxic ischemic encephalopathy

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Zhao Xiang; Ma Jiwei; Wu Lide

2004-01-01

Objective: To explore CT characteristics of hypoxic ischemic encephalopathy (HIE), and to improve the accuracy of CT diagnosis. Methods: 50 cases of neonatal asphyxia in perinatal period diagnosed as hypoxic ischemic encephalopathy by CT was analyzed. Results: The main manifestation of hypoxic ischemic encephalopathy is cerebral edema and intracranial hemorrhage. Focal or diffuse hypo-dense lesion and hyper-dense area in various location and morphology were seen on CT images. (1) Localized diffuse hypo-dense area in 1 or 2 cerebral lobe were found in 17 cases, and the lesions were localized in frontal lobe (n=6), in frontotemporal lobe (n=5), and in temporo-occipital lobe (n=6). (2) Hypo-density region involving more than three cerebral lobes were found in 18 cases, and abnormalities were found in frontotemporal and parietal lobe (n=8), accompanying with subarachnoid hemorrhage (n=2); in frontal, temporal and occipital lobe (n=6), in which cerebral hemorrhage was complicated (n=1); and in other cerebral lobe (n=4). (3) Diffuse low-density region in all cerebral lobe were found in 15 cases, in which subarachnoid hemorrhage was complicated in 4 cases, and ventricular hemorrhage was found in 2 case. Conclusion: CT imaging plays an important role in diagnosis of hypoxic ischemic encephalopathy and has shown its clinical value

Apparent diffusion coefficient histogram analysis of neonatal hypoxic-ischemic encephalopathy

International Nuclear Information System (INIS)

Cauley, Keith A.; Filippi, Christopher G.

2014-01-01

Diffusion-weighted imaging is a valuable tool in the assessment of the neonatal brain, and changes in diffusion are seen in normal development as well as in pathological states such as hypoxic-ischemic encephalopathy (HIE). Various methods of quantitative assessment of diffusion values have been reported. Global ischemic injury occurring during the time of rapid developmental changes in brain myelination can complicate the imaging diagnosis of neonatal HIE. To compare a quantitative method of histographic analysis of brain apparent coefficient (ADC) maps to the qualitative interpretation of routine brain MR imaging studies. We correlate changes in diffusion values with gestational age in radiographically normal neonates, and we investigate the sensitivity of the method as a quantitative measure of hypoxic-ischemic encephalopathy. We reviewed all brain MRI studies from the neonatal intensive care unit (NICU) at our university medical center over a 4-year period to identify cases that were radiographically normal (23 cases) and those with diffuse, global hypoxic-ischemic encephalopathy (12 cases). We histographically displayed ADC values of a single brain slice at the level of the basal ganglia and correlated peak (s-sD av ) and lowest histogram values (s-sD lowest ) with gestational age. Normative s-sD av values correlated significantly with gestational age and declined linearly through the neonatal period (r 2 = 0.477, P av and s-sD lowest ADC values than were reflected in the normative distribution; several cases of HIE fell within a 95% confidence interval for normative studies, and one case demonstrated higher-than-normal s-sD av . Single-slice histographic display of ADC values is a rapid and clinically feasible method of quantitative analysis of diffusion. In this study normative values derived from consecutive neonates without radiographic evidence of ischemic injury are correlated with gestational age, declining linearly throughout the perinatal period. This

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

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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.

Hypoxic training methods for improving endurance exercise performance

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Jacob A. Sinex

2015-12-01

Full Text Available Endurance athletic performance is highly related to a number of factors that can be altered through altitude and hypoxic training including increases in erythrocyte volume, maximal aerobic exercise capacity, capillary density, and economy. Physiological adaptations in response to acute and chronic exposure to hypoxic environments are well documented and range from short-term detrimental effects to longer-term adaptations that can improve performance at altitude and in sea-level competitions. Many altitude and hypoxic training protocols have been developed, employing various combinations of living and training at sea-level, low, moderate, and high altitudes and utilizing natural and artificial altitudes, with varying degrees of effectiveness. Several factors have been identified that are associated with individual responses to hypoxic training, and techniques for identifying those athletes most likely to benefit from hypoxic training continue to be investigated. Exposure to sufficiently high altitude (2000–3000 m for more than 12 h/day, while training at lower altitudes, for a minimum of 21 days is recommended. Timing of altitude training related to competition remains under debate, although general recommendations can be considered.

Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

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Thomas R. Cox

2015-12-01

Full Text Available The cancer secretome includes all of the macromolecules secreted by cells into their microenvironment. Cancer cell secretomes are significantly different to that of normal cells reflecting the changes that normal cells have undergone during their transition to malignancy. More importantly, cancer secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397 and the complete proteomic, bioinformatic and biological analyses are reported in Cox et al. (2015 [1].

[Follow-up of newborns with hypoxic-ischaemic encephalopathy].

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Martínez-Biarge, M; Blanco, D; García-Alix, A; Salas, S

2014-07-01

Hypothermia treatment for newborn infants with hypoxic-ischemic encephalopathy reduces the number of neonates who die or have permanent neurological deficits. Although this therapy is now standard of care, neonatal hypoxic-ischaemic encephalopathy still has a significant impact on the child's neurodevelopment and quality of life. Infants with hypoxic-ischaemic encephalopathy should be enrolled in multidisciplinary follow-up programs in order to detect impairments, to initiate early intervention, and to provide counselling and support for families. This article describes the main neurodevelopmental outcomes after term neonatal hypoxic-ischaemic encephalopathy. We offer recommendations for follow-up based on the infant's clinical condition and other prognostic indicators, mainly neonatal neuroimaging. Other aspects, such as palliative care and medico-legal issues, are also briefly discussed. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Plasticity in the Neonatal Brain following Hypoxic-Ischaemic Injury

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Eridan Rocha-Ferreira

2016-01-01

Full Text Available Hypoxic-ischaemic damage to the developing brain is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy, and cognitive disabilities. The developmental stage of the brain and the severity of the insult influence the selective regional vulnerability and the subsequent clinical manifestations. The increased susceptibility to hypoxia-ischaemia (HI of periventricular white matter in preterm infants predisposes the immature brain to motor, cognitive, and sensory deficits, with cognitive impairment associated with earlier gestational age. In term infants HI causes selective damage to sensorimotor cortex, basal ganglia, thalamus, and brain stem. Even though the immature brain is more malleable to external stimuli compared to the adult one, a hypoxic-ischaemic event to the neonate interrupts the shaping of central motor pathways and can affect normal developmental plasticity through altering neurotransmission, changes in cellular signalling, neural connectivity and function, wrong targeted innervation, and interruption of developmental apoptosis. Models of neonatal HI demonstrate three morphologically different types of cell death, that is, apoptosis, necrosis, and autophagy, which crosstalk and can exist as a continuum in the same cell. In the present review we discuss the mechanisms of HI injury to the immature brain and the way they affect plasticity.

Vasotrophic Regulation of Age-Dependent Hypoxic Cerebrovascular Remodeling

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Silpanisong, Jinjutha; Pearce, William J.

2015-01-01

Hypoxia can induce functional and structural vascular remodeling by changing the expression of trophic factors to promote homeostasis. While most experimental approaches have been focused on functional remodeling, structural remodeling can reflect changes in the abundance and organization of vascular proteins that determine functional remodeling. Better understanding of age-dependent hypoxic macrovascular remodeling processes of the cerebral vasculature and its clinical implications require knowledge of the vasotrophic factors that influence arterial structure and function. Hypoxia can affect the expression of transcription factors, classical receptor tyrosine kinase factors, non-classical G-protein coupled factors, catecholamines, and purines. Hypoxia’s remodeling effects can be mediated by Hypoxia Inducible Factor (HIF) upregulation in most vascular beds, but alterations in the expression of growth factors can also be independent of HIF. PPARγ is another transcription factor involved in hypoxic remodeling. Expression of classical receptor tyrosine kinase ligands, including vascular endothelial growth factor, platelet derived growth factor, fibroblast growth factor and angiopoietins, can be altered by hypoxia which can act simultaneously to affect remodeling. Tyrosine kinase-independent factors, such as transforming growth factor, nitric oxide, endothelin, angiotensin II, catecholamines, and purines also participate in the remodeling process. This adaptation to hypoxic stress can fundamentally change with age, resulting in different responses between fetuses and adults. Overall, these mechanisms integrate to assure that blood flow and metabolic demand are closely matched in all vascular beds and emphasize the view that the vascular wall is a highly dynamic and heterogeneous tissue with multiple cell types undergoing regular phenotypic transformation. PMID:24063376

Studies on the toxicity of RSU-1069

International Nuclear Information System (INIS)

Whitmore, G.F.; Gulyas, S.

1986-01-01

RSU-1069 combines an aziridine function with a 2-nitroimidazole and has been reported to exhibit extraordinary radiosensitization both in vitro and in vivo. Such sensitization appears to be at variance with the electron affinity of the compound. In addition, recent experiments suggest that the compound is highly toxic to hypoxic tumor cells in vivo. On the assumption that the observed radiosensitizing ability may be a manifestation of toxicity and because of the high in vivo toxicity, we have investigated aerobic and hypoxic toxicity, both in wild type CHO cells and in mutants sensitive to a variety of DNA damaging agents. With wild type cells under aerobic conditions, the compound is approximately 50 times as toxic as misonidazole and under hypoxic conditions, approximately 250 times as toxic. The ratio of hypoxic to aerobic toxicity is approximately 80 times. Under aerobic conditions, repair-deficient mutants are 10 times as sensitive to RSU-1069 as wild type cells and approximately 100 times as sensitive under hypoxic conditions. The ratio of hypoxic to aerobic toxicity for the mutant cells is approximately 900. Based on these observations, we suggest that under aerobic conditions the aziridine function is primarily responsible for toxicity, whereas, under hypoxic conditions, the aziridine moiety combined with a reduced 2-nitroimidazole moiety produces a bifunctional agent

Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit

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Cukrov, Dubravka; Zermiani, Monica; Brizzolara, Stefano; Cestaro, Alessandro; Licausi, Francesco; Luchinat, Claudio; Santucci, Claudio; Tenori, Leonardo; Van Veen, Hans; Zuccolo, Andrea; Ruperti, Benedetto; Tonutti, Pietro

2016-01-01

The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest

Hypoxic stress-induced changes in ribosomes of maize seedling roots

International Nuclear Information System (INIS)

Bailey-Serres, J.; Freeling, M.

1990-01-01

The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with [ 35 S]methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with [ 32 P]orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy

International Nuclear Information System (INIS)

Biaglow, J.E.; Varnes, M.E.; Epp, E.R.; Clark, E.P.

1984-01-01

The rate of GSH resynthesis has been measured in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The authors suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased

Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

Science.gov (United States)

Rigo, Federica; De Stefano, Nicola; Navarro-Tableros, Victor; David, Ezio; Rizza, Giorgia; Catalano, Giorgia; Gilbo, Nicholas; Maione, Francesca; Gonella, Federica; Roggio, Dorotea; Martini, Silvia; Patrono, Damiano; Salizzoni, Mauro; Camussi, Giovanni; Romagnoli, Renato

2018-05-01

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV). We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction. During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014). HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

Application of altitude/hypoxic training by elite athletes.

Science.gov (United States)

Wilber, Randall L

2007-09-01

At the Olympic level, differences in performance are typically less than 0.5%. This helps explain why many contemporary elite endurance athletes in summer and winter sport incorporate some form of altitude/hypoxic training within their year-round training plan, believing that it will provide the "competitive edge" to succeed at the Olympic level. The purpose of this paper is to describe the practical application of altitude/hypoxic training as used by elite athletes. Within the general framework of the paper, both anecdotal and scientific evidence will be presented relative to the efficacy of several contemporary altitude/hypoxic training models and devices currently used by Olympic-level athletes for the purpose of legally enhancing performance. These include the three primary altitude/hypoxic training models: 1) live high+train high (LH+TH), 2) live high+train low (LH+TL), and 3) live low+train high (LL+TH). The LH+TL model will be examined in detail and will include its various modifications: natural/terrestrial altitude, simulated altitude via nitrogen dilution or oxygen filtration, and hypobaric normoxia via supplemental oxygen. A somewhat opposite approach to LH+TL is the altitude/hypoxic training strategy of LL+TH, and data regarding its efficacy will be presented. Recently, several of these altitude/hypoxic training strategies and devices underwent critical review by the World Anti-Doping Agency (WADA) for the purpose of potentially banning them as illegal performance-enhancing substances/methods. This paper will conclude with an update on the most recent statement from WADA regarding the use of simulated altitude devices.

HDAC6 maintains mitochondrial connectivity under hypoxic stress by suppressing MARCH5/MITOL dependent MFN2 degradation

International Nuclear Information System (INIS)

Kim, Hak-June; Nagano, Yoshito; Choi, Su Jin; Park, Song Yi; Kim, Hongtae; Yao, Tso-Pang; Lee, Joo-Yong

2015-01-01

Mitochondria undergo fusion and fission in response to various metabolic stresses. Growing evidences have suggested that the morphological change of mitochondria by fusion and fission plays a critical role in protecting mitochondria from metabolic stresses. Here, we showed that hypoxia treatment could induce interaction between HDAC6 and MFN2, thus protecting mitochondrial connectivity. Mechanistically, we demonstrated that a mitochondrial ubiquitin ligase MARCH5/MITOL was responsible for hypoxia-induced MFN2 degradation in HDAC6 deficient cells. Notably, genetic abolition of HDAC6 in amyotrophic lateral sclerosis model mice showed MFN2 degradation with MARCH5 induction. Our results indicate that HDAC6 is a critical regulator of MFN2 degradation by MARCH5, thus protecting mitochondrial connectivity from hypoxic stress. - Highlights: • Hypoxic stress induces the interaction between HDAC6 and MFN2. • Hypoxic stress activates MARCH5 in HDAC6 deficient cells to degrade MFN2. • HDAC6 is required to maintain mitochondrial connectivity under hypoxia. • MARCH5 is increased and promotes the degradation of MFN2 in HDAC6 KO ALS mice

HDAC6 maintains mitochondrial connectivity under hypoxic stress by suppressing MARCH5/MITOL dependent MFN2 degradation

Energy Technology Data Exchange (ETDEWEB)

Kim, Hak-June [Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Nagano, Yoshito [Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, 734-8551 (Japan); Choi, Su Jin; Park, Song Yi [Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Kim, Hongtae [Department of Biological Sciences, Sungkyunkwan University (SKKU), Suwon, 440-746 (Korea, Republic of); Yao, Tso-Pang, E-mail: tsopang.yao@duke.edu [Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710 (United States); Lee, Joo-Yong, E-mail: leejooyong@cnu.ac.kr [Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of)

2015-09-04

Mitochondria undergo fusion and fission in response to various metabolic stresses. Growing evidences have suggested that the morphological change of mitochondria by fusion and fission plays a critical role in protecting mitochondria from metabolic stresses. Here, we showed that hypoxia treatment could induce interaction between HDAC6 and MFN2, thus protecting mitochondrial connectivity. Mechanistically, we demonstrated that a mitochondrial ubiquitin ligase MARCH5/MITOL was responsible for hypoxia-induced MFN2 degradation in HDAC6 deficient cells. Notably, genetic abolition of HDAC6 in amyotrophic lateral sclerosis model mice showed MFN2 degradation with MARCH5 induction. Our results indicate that HDAC6 is a critical regulator of MFN2 degradation by MARCH5, thus protecting mitochondrial connectivity from hypoxic stress. - Highlights: • Hypoxic stress induces the interaction between HDAC6 and MFN2. • Hypoxic stress activates MARCH5 in HDAC6 deficient cells to degrade MFN2. • HDAC6 is required to maintain mitochondrial connectivity under hypoxia. • MARCH5 is increased and promotes the degradation of MFN2 in HDAC6 KO ALS mice.

Silencing of Taxol-Sensitizer Genes in Cancer Cells: Lack of Sensitization Effects

International Nuclear Information System (INIS)

Huang, Shang-Lang; Chao, Chuck C.-K.

2015-01-01

A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug

Silencing of Taxol-Sensitizer Genes in Cancer Cells: Lack of Sensitization Effects

Energy Technology Data Exchange (ETDEWEB)

Huang, Shang-Lang [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chao, Chuck C.-K., E-mail: cckchao@mail.cgu.edu.tw [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Department of Medical Research and Development, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan (China)

2015-06-16

A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.

Irradiation of mammalian cells in the presence of diamide and low concentrations of oxygen at conventional and at ultrahigh dose rates

International Nuclear Information System (INIS)

Clark, E.P.; Michaels, H.B.; Peterson, E.C.; Epp, E.R.

1983-01-01

The response of cultured CHO cells to ultrahigh-dose-radiation (approx.10 9 Gy/sec) has been previously studied extensively using the thin-layer cell-handling technique developed in this laboratory. When the cells are equilibrated with a low concentration of oxygen, e.g., 0.44% O 2 , a breaking survival curve, due to radiolytic depletion of the oxygen, is observed. Hypoxic cells irradiated in the presence of the nitroimidazoles (e.g., misonidazole) are sensitized at ultrahigh dose rates in a dose-modifying manner, similar to that observed at conventional dose rates. These radiosensitizer compounds, if present in cells equilibrated with a low concentration of oxygen, prevent the breaking behavior of the survival curve, an observation believed to be due to the sensitizer interfering with the oxygen depletion process, leaving oxygen free to sensitize. Such experiments have recently been extended to studies with diamide, which, unlike the other sensitizers tested, acts primarily as a shoulder-modifying rather than a dose-modifying agent in hypoxic mammalian cells. These data indicate that diamide is active as a sensitizer at ultrahigh dose rates in a manner similar to that observed at conventional dose rates, and does modify the shape of the breaking survival curve observed with low concentrations of oxygen

Strategies To Assess Hypoxic/HIF-1-Active Cancer Cells for the Development of Innovative Radiation Therapy

International Nuclear Information System (INIS)

Yeom, Chan Joo; Zeng, Lihua; Zhu, Yuxi; Hiraoka, Masahiro; Harada, Hiroshi

2011-01-01

Local tumor recurrence and distant tumor metastasis frequently occur after radiation therapy and result in the death of cancer patients. These problems are caused, at least in part, by a tumor-specific oxygen-poor microenvironment, hypoxia. Oxygen-deprivation is known to inhibit the chemical ionization of both intracellular macro-molecules and water, etc., and thus reduce the cytotoxic effects of radiation. Moreover, DNA damage produced by free radicals is known to be more repairable under hypoxia than normoxia. Hypoxia is also known to induce biological tumor radioresistance through the activation of a transcription factor, hypoxia-inducible factor 1 (HIF-1). Several potential strategies have been devised in radiation therapy to overcome these problems; however, they have not yet achieved a complete remission. It is essential to reveal the intratumoral localization and dynamics of hypoxic/HIF-1-active tumor cells during tumor growth and after radiation therapy, then exploit the information to develop innovative therapeutic strategies, and finally damage radioresistant cells. In this review, we overview problems caused by hypoxia/HIF-1-active cells in radiation therapy for cancer and introduce strategies to assess intratumoral hypoxia/HIF-1 activity

Strategies To Assess Hypoxic/HIF-1-Active Cancer Cells for the Development of Innovative Radiation Therapy

Energy Technology Data Exchange (ETDEWEB)

Yeom, Chan Joo [Group of Radiation and Tumor Biology, Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Zeng, Lihua; Zhu, Yuxi [Group of Radiation and Tumor Biology, Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Department of Radiation Oncology and Image-applied Therapy, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Hiraoka, Masahiro [Department of Radiation Oncology and Image-applied Therapy, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Harada, Hiroshi, E-mail: hharada@kuhp.kyoto-u.ac.jp [Group of Radiation and Tumor Biology, Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan)

2011-09-15

Local tumor recurrence and distant tumor metastasis frequently occur after radiation therapy and result in the death of cancer patients. These problems are caused, at least in part, by a tumor-specific oxygen-poor microenvironment, hypoxia. Oxygen-deprivation is known to inhibit the chemical ionization of both intracellular macro-molecules and water, etc., and thus reduce the cytotoxic effects of radiation. Moreover, DNA damage produced by free radicals is known to be more repairable under hypoxia than normoxia. Hypoxia is also known to induce biological tumor radioresistance through the activation of a transcription factor, hypoxia-inducible factor 1 (HIF-1). Several potential strategies have been devised in radiation therapy to overcome these problems; however, they have not yet achieved a complete remission. It is essential to reveal the intratumoral localization and dynamics of hypoxic/HIF-1-active tumor cells during tumor growth and after radiation therapy, then exploit the information to develop innovative therapeutic strategies, and finally damage radioresistant cells. In this review, we overview problems caused by hypoxia/HIF-1-active cells in radiation therapy for cancer and introduce strategies to assess intratumoral hypoxia/HIF-1 activity.

Mechanism of action of shoulder-modifying anoxic radiosensitizers and their use in combination with other treatment modalities. Coordinated programme on improvement in radiotherapy of cancer using modifiers of radiosensitivity of cells

International Nuclear Information System (INIS)

Harris, J.W.

1982-01-01

Research during the period of the Research Agreement has focused on four approaches to increased killing of tumor cells: hypoxic sensitizers, protective agents, hyperthermia and chemotherapeutic drugs. Various considerations have been examined, both in vitro and, to a lesser extent, in vivo, but the chief emphasis has been on the basic mechanisms involved. Results have been published in more than twenty publications and may be summarized as follows. Chemical sensitization: Results with the glutathione-oxidant ''diamide'' confirmed the importance of cellular GSH in the oxygen effect. Moreover, the possibility of combining agents like diamide with the nitroimidazole agents (e.g. misonidazole) was examined. Perhaps the most fundamental observation was the decrease in survival curve shoulder seen, for hypoxic cells, when irradiation was done in the presence of diamide. Chemical protection: The thio-phosphate WR2721, with respect to protection of various tissues and to the basis for its differential protection of normal tissues vs tumors was tested. Other studies have examined the mechanism(s) by which thiols protect against X-ray damage and the effects of certain chemotherapy drugs (e.g. Cis-platinum). Hyperthermia: Heat has been studied in conjunction with neutron irradiation (there is no differential effect on hypoxic cells) and at lower temperatures (40 0 ). The latter treatment (40 0 C for 2 hrs) removes the shoulder from the euoxic X-ray survival curve. Chemotherapy agents: These studies have explored the effects of clinically-used drugs on euoxic and hypoxic cells. No differences in efficiency are seen for acutely hypoxic cells. A system has been developed for chronic hypoxic in vitro and here different drug effects may be seen. Of particular interest is the decrease in OER observed in chronically hypoxic cells, possibly due to a decreased cellular content of glutathione. The damage due to Cis-platinum can be protected against, in large measure, by cysteamine

Sulphonylurea drugs reduce hypoxic damage in the isolated perfused rat kidney.

Science.gov (United States)

Engbersen, R; Moons, M M; Wouterse, A C; Dijkman, H B; Kramers, C; Smits, P; Russel, F G

2000-08-01

Sulphonylurea drugs have been shown to protect against hypoxic damage in isolated proximal tubules of the kidney. In the present study we investigated whether these drugs can protect against hypoxic damage in a whole kidney preparation. Tolbutamide (200 microM) and glibenclamide (10 microM) were applied to the isolated perfused rat kidney prior to changing the gassing from oxygen to nitrogen for 30 min. Hypoxic perfusions resulted in an increased fractional excretion of glucose (FE % glucose 14.3+/-1.5 for hypoxic perfusions vs 4.9+/-1.6 for normoxic perfusions, mean +/- s.e. mean, P<0.05), which could be completely restored by 200 microM tolbutamide (5.7+/-0.4 for tolbutamide vs 14.3+/-1.5 for untreated hypoxic kidneys, P<0.01). Furthermore, tolbutamide reduced the total amount of LDH excreted in the urine (220+/-100 mU for tolbutamide vs. 1220+/-160 mU for untreated hypoxic kidneys, P<0.01). Comparable results were obtained with glibenclamide (10 microM). In agreement with the effect on functional parameters, ultrastructural analysis of proximal tubules showed increased brush border preservation in tolbutamide treated kidneys compared to untreated hypoxic kidneys. We conclude that glibenclamide and tolbutamide are both able to reduce hypoxic damage to proximal tubules in the isolated perfused rat kidney when applied in the appropriate concentrations.

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

Science.gov (United States)

Wardman, Peter

2018-03-20

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

Fast kinetics of the oxygen effect in irradiated mammalian cells

International Nuclear Information System (INIS)

Watts, M.E.; Maughan, R.L.; Michael, B.D.

1978-01-01

A technique using a fast gas transfer with a single pulse of electrons (the gas-explosion technique) has been used to investigate the time-dependence of the dose-modifying action of oxygen in irradiated V79 Chinese hamster cells. Oxygen did not significantly alter the shapes of the survival curves. The dose-modifying factor between the fully oxic and fully hypoxic (oxygen at 9000 ms) curve was 2.6. The dose-modifying factor for the survival curve drawn for oxygen contact at 0.3 ms after irradiation was 1.5 relative to the hypoxic curve. The duration of the post-effect (oxygen contact after irradiation) indicated that oxygen-dependent damage has a lifetime extending into the ms time-range. In the pre-effect time region (oxygen contact before irradiation) 1 to 2 ms oxygen contact was required to achieve the full sensitization. The results are discussed with reference to the diffusion time for oxygen to reach the sensitive site within the cell. (U.K.)

Hypoxia-inducible factor-1β (HIF-1β) is upregulated in a HIF-1α-dependent manner in 518A2 human melanoma cells under hypoxic conditions

Energy Technology Data Exchange (ETDEWEB)

Mandl, Markus, E-mail: mmandl@mail.austria.com; Kapeller, Barbara; Lieber, Roman; Macfelda, Karin

2013-04-26

Highlights: •HIF-1β is a hypoxia-responsive protein in 518A2 human melanoma cells. •HIF-1β is upregulated in a HIF-1α-dependent manner under hypoxic conditions. •HIF-1β is not elevated due to heterodimerization with HIF-1α per se. •HIF-1β inducibility has a biological relevance as judged in Het-CAM model. -- Abstract: Solid tumors include hypoxic areas due to excessive cell proliferation. Adaptation to low oxygen levels is mediated by the hypoxia-inducible factor (HIF) pathway promoting invasion, metastasis, metabolic alterations, chemo-resistance and angiogenesis. The transcription factor HIF-1, the major player within this pathway consists of HIF-1α and HIF-1β. The alpha subunit is continuously degraded under normoxia and becomes stabilized under reduced oxygen supply. In contrast, HIF-1β is generally regarded as constitutively expressed and being present in excess within the cell. However, there is evidence that the expression of this subunit is more complex. The aim of this study was to investigate the role of HIF-1β in human melanoma cells. Among a panel of five different cell lines, in 518A2 cells exposed to the hypoxia-mimetic cobalt chloride HIF-1β was rapidly elevated on protein level. Knockdown experiments performed under cobalt chloride-exposure and hypoxia revealed that this effect was mediated by HIF-1α. The non-canonical relationship between these subunits was further confirmed by pharmacologic inhibition of HIF-1α and by expression of a dominant-negative HIF mutant. Overexpression of HIF-1α showed a time delay in HIF-1β induction, thus arguing for HIF-1β de novo synthesis rather than protein stabilization by heterodimerization. A Hen’s egg test-chorioallantoic membrane model of angiogenesis and invasion indicated a local expression of HIF-1β and implies a biological relevance of these findings. In summary, this study demonstrates the HIF-1α-dependent regulation of HIF-1β under hypoxic conditions for the first time. The

Hypoxia-inducible factor-1β (HIF-1β) is upregulated in a HIF-1α-dependent manner in 518A2 human melanoma cells under hypoxic conditions

International Nuclear Information System (INIS)

Mandl, Markus; Kapeller, Barbara; Lieber, Roman; Macfelda, Karin

2013-01-01

Highlights: •HIF-1β is a hypoxia-responsive protein in 518A2 human melanoma cells. •HIF-1β is upregulated in a HIF-1α-dependent manner under hypoxic conditions. •HIF-1β is not elevated due to heterodimerization with HIF-1α per se. •HIF-1β inducibility has a biological relevance as judged in Het-CAM model. -- Abstract: Solid tumors include hypoxic areas due to excessive cell proliferation. Adaptation to low oxygen levels is mediated by the hypoxia-inducible factor (HIF) pathway promoting invasion, metastasis, metabolic alterations, chemo-resistance and angiogenesis. The transcription factor HIF-1, the major player within this pathway consists of HIF-1α and HIF-1β. The alpha subunit is continuously degraded under normoxia and becomes stabilized under reduced oxygen supply. In contrast, HIF-1β is generally regarded as constitutively expressed and being present in excess within the cell. However, there is evidence that the expression of this subunit is more complex. The aim of this study was to investigate the role of HIF-1β in human melanoma cells. Among a panel of five different cell lines, in 518A2 cells exposed to the hypoxia-mimetic cobalt chloride HIF-1β was rapidly elevated on protein level. Knockdown experiments performed under cobalt chloride-exposure and hypoxia revealed that this effect was mediated by HIF-1α. The non-canonical relationship between these subunits was further confirmed by pharmacologic inhibition of HIF-1α and by expression of a dominant-negative HIF mutant. Overexpression of HIF-1α showed a time delay in HIF-1β induction, thus arguing for HIF-1β de novo synthesis rather than protein stabilization by heterodimerization. A Hen’s egg test-chorioallantoic membrane model of angiogenesis and invasion indicated a local expression of HIF-1β and implies a biological relevance of these findings. In summary, this study demonstrates the HIF-1α-dependent regulation of HIF-1β under hypoxic conditions for the first time. The

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device

Energy Technology Data Exchange (ETDEWEB)

Germain, Todd; Ansari, Megan; Pappas, Dimitri, E-mail: d.pappas@ttu.edu

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. - Highlights: • Microfluidic system switches rapidly between normoxia and hypoxia (5 min). • Observation of rapid adaptation of PC3 cells to hypoxia and normoxia (30 min). • Drug susceptibility in tumor cells restored after chip switched to normoxia for 30 min.

Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

DEFF Research Database (Denmark)

Cox, T.R.; Schoof, Erwin; Gartland, A.

2015-01-01

secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic...... and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397...

Neuro-overprotection? A functional evaluation of clomethiazole-induced neuroprotection following hypoxic-ischemic injury.

Science.gov (United States)

Gilby, K L; Kelly, M E; McIntyre, D C; Robertson, H A

2005-01-01

Hypoxic-ischemic (H-I) injury produces extensive damage to the hippocampus of young rats. We have recently shown that administration of 125 mg kg-1 clomethiazole (CMZ), a GABA(A)-agonist, provides complete histological protection against H-I injury if administered 3 h post-H-I (Brain Res 1035 (2005) 194). However, whether that histological protection translates into lasting functional preservation is unclear. To determine whether hippocampal-based circuits remain functionally intact in CMZ-protected H-I rats, we administered 125 mg kg-1 (high dose [CMZ-HD]) or 65 mg kg-1 (low dose [CMZ-LD]) CMZ, 3 h post-H-I, and examined numerous kindling parameters in the dorsal hippocampus 60 days following H-I. Kindling parameters included afterdischarge (AD) thresholds (ADTs), AD durations and kindling rates. Additional groups assessed included vehicle-injected H-I (VIH), hypoxic, ligated and naive rats. VIH, CMZ-HD, CMZ-LD and hypoxic rats all exhibited significantly faster kindling rates than naive rats. Thus, a previous traumatic event, even hypoxia alone, facilitated subsequent seizure propagation. Still, a significantly slower kindling rate was evident in CMZ-HD rats than in hypoxic, VIH or CMZ-LD rats. Moreover, while longer pre-kindling AD durations were observed in the damaged hippocampus of VIH compared with naive rats, this was not true for either CMZ-treated groups, hypoxic or ligated rats. Collectively, these findings suggest CMZ can suppress the epileptogenic effects of H-I. Surprisingly, however, both groups of CMZ-treated rats exhibited a four to nine times greater ADT than any other group and this effect was most profound in the CMZ-protected hippocampus. Thus, CMZ administration protected local neurons against terminal insult and left network excitability relatively normal with respect to seizure offset mechanisms but also caused profound elevation of local ADTs, which suggests a local hypoexcitability/increased inhibition. Finally, this study demonstrates

Hypothermia therapy for newborns with hypoxic ischemic encephalopathy.

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Silveira, Rita C; Procianoy, Renato S

2015-01-01

Therapeutic hypothermia reduces cerebral injury and improves the neurological outcome secondary to hypoxic ischemic encephalopathy in newborns. It has been indicated for asphyxiated full-term or near-term newborn infants with clinical signs of hypoxic-ischemic encephalopathy (HIE). A search was performed for articles on therapeutic hypothermia in newborns with perinatal asphyxia in PubMed; the authors chose those considered most significant. There are two therapeutic hypothermia methods: selective head cooling and total body cooling. The target body temperature is 34.5 °C for selective head cooling and 33.5 °C for total body cooling. Temperatures lower than 32 °C are less neuroprotective, and temperatures below 30 °C are very dangerous, with severe complications. Therapeutic hypothermia must start within the first 6h after birth, as studies have shown that this represents the therapeutic window for the hypoxic-ischemic event. Therapy must be maintained for 72 h, with very strict control of the newborn's body temperature. It has been shown that therapeutic hypothermia is effective in reducing neurologic impairment, especially in full-term or near-term newborns with moderate hypoxic-ischemic encephalopathy. Therapeutic hypothermia is a neuroprotective technique indicated for newborn infants with perinatal asphyxia and hypoxic-ischemic encephalopathy. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

The metal-responsive transcription factor-1 contributes to HIF-1 activation during hypoxic stress

International Nuclear Information System (INIS)

Murphy, Brian J.; Sato, Barbara G.; Dalton, Timothy P.; Laderoute, Keith R.

2005-01-01

Hypoxia-inducible factor-1 (HIF-1), the major transcriptional regulator of the mammalian cellular response to low oxygen (hypoxia), is embedded within a complex network of signaling pathways. We have been investigating the importance of another stress-responsive transcription factor, MTF-1, for the adaptation of cells to hypoxia. This article reports that MTF-1 plays a central role in hypoxic cells by contributing to HIF-1 activity. Loss of MTF-1 in transformed Mtf1 null mouse embryonic fibroblasts (MEFs) results in an attenuation of nuclear HIF-1α protein accumulation, HIF-1 transcriptional activity, and expression of an established HIF-1 target gene, glucose transporter-1 (Glut1). Mtf1 null (Mtf1 KO) MEFs also have constitutively higher levels of both glutathione (GSH) and the rate-limiting enzyme involved in GSH synthesis-glutamate cysteine ligase catalytic subunit-than wild type cells. The altered cellular redox state arising from increased GSH may perturb oxygen-sensing mechanisms in hypoxic Mtf1 KO cells and decrease the accumulation of HIF-1α protein. Together, these novel findings define a role for MTF-1 in the regulation of HIF-1 activity

A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

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Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

2014-02-28

NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. Copyright © 2014 Elsevier Inc. All rights reserved.

Hypoxic-induced stress protein expression in rat cardiac myocytes

International Nuclear Information System (INIS)

Howard, G.; Geoghegan, T.E.

1986-01-01

Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished O 2 supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95 kDa proteins increase with time of exposure to hypoxia, while the mRNA coding for the 71 kDa protein is transiently induced. The authors extended these studies to investigate the expression of stress proteins in isolated rat cardiac myocytes. Freshly prepared myocytes were exposed to control, hypoxic, anoxic, or heat-shock environments for up to 16 h. The proteins were then labeled for 6 hours with [ 35 S]methionine. Analysis of the solubilized proteins by SDS-PAGE and autoradiography showed that there was a 6-fold increase in synthesis of the 85 kDa protein upon exposure to hypoxia but not heat-shock conditions. The 71 kDa protein was present at high levels in both control and treated myocyte protein preparations, and presumably had been induced during the isolation procedure. Total RNA isolated from intact rat heart and isolated myocytes was compared by cell-free translation analysis and showed induction of RNAs coding for several stress proteins in the myocyte preparation. The induced proteins at 85 and 95 kDa have molecular weights similar to reported cell stress and/or glucose-regulated proteins

Bifurcate effects of glucose on caspase-independent cell death during hypoxia

International Nuclear Information System (INIS)

Aki, Toshihiko; Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi

2010-01-01

We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

Oxygen Generating Biomaterials Preserve Skeletal Muscle Homeostasis under Hypoxic and Ischemic Conditions

Science.gov (United States)

2013-08-26

injection” protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up...Oxygen Generating Biomaterials Preserve Skeletal Muscle Homeostasis under Hypoxic and Ischemic Conditions Catherine L. Ward, Benjamin T. Corona...investigation was to determine if sodium percarbonate (SPO), an oxygen generating biomaterial, is capable of maintaining resting skeletal muscle

Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins.

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Gogate, Shilpa S; Nasser, Rena; Shapiro, Irving M; Risbud, Makarand V

2011-07-01

To determine whether hypoxia and hypoxia-inducible factor (HIF) proteins regulate expression of β-1,3-glucuronyltransferase 1 (GlcAT-1), a key enzyme in glycosaminoglycan synthesis in nucleus pulposus cells. Real-time reverse transcriptase-polymerase chain reaction and Western blotting were used to measure GlcAT-1 expression. Transfections were performed to determine the effect of HIF-1α and HIF-2α on GlcAT-1 promoter activity. Under hypoxic conditions there was an increase in GlcAT-1 expression; a significant increase in promoter activity was seen both in nucleus pulposus cells and in N1511 chondrocytes. We investigated whether HIF controlled GlcAT-1 expression. Suppression of HIF-1α and HIF-2α induced GlcAT-1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA-HIF-1α as well as with CA-HIF-2α suppressed GlcAT-1 promoter activity only in nucleus pulposus cells, suggesting a cell type-specific regulation. Site-directed mutagenesis and deletion constructs were used to further confirm the suppressive effect of HIFs on GlcAT-1 promoter function in nucleus pulposus cells. Although it was evident that interaction of HIF with hypoxia-responsive elements resulted in suppression of basal promoter activity, it was not necessary for transcriptional suppression. This result suggested both a direct and an indirect mode of regulation, possibly through recruitment of a HIF-dependent repressor. Finally, we showed that hypoxic expression of GlcAT-1 was also partially dependent on MAPK signaling. These studies demonstrate that hypoxia regulates GlcAT-1 expression through a signaling network comprising both activator and suppressor molecules, and that this regulation is unique to nucleus pulposus cells. Copyright © 2011 by the American College of Rheumatology.

Hypoxic conditioning as a new therapeutic modality

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Samuel eVerges

2015-06-01

Full Text Available Preconditioning refers to a procedure by which a single noxious stimulus below the threshold of damage is applied to the tissue in order to increase resistance to the same or even different noxious stimuli given above the threshold of damage. Hypoxic preconditioning relies on complex and active defenses that organisms have developed to counter the adverse consequences of oxygen deprivation. The protection it confers against ischemic attack for instance as well as the underlying biological mechanisms have been extensively investigated in animal models. Based on these data, hypoxic conditioning (consisting in recurrent exposure to hypoxia has been suggested a potential non-pharmacological therapeutic intervention to enhance some physiological functions in individuals in whom acute or chronic pathological events are anticipated or existing. In addition to healthy subjects, some benefits have been reported in patients with cardiovascular and pulmonary diseases as well as in overweight and obese individuals. Hypoxic conditioning consisting in sessions of intermittent exposure to moderate hypoxia repeated over several weeks may induce hematological, vascular, metabolic and neurological effects. This review addresses the existing evidence regarding the use of hypoxic conditioning as a potential therapeutic modality and emphasizes on many remaining issues to clarify and future researches to be performed in the field.

Perinatal Hypoxic-Ischemic brain injury; MR findings

International Nuclear Information System (INIS)

Park, Dong Woo; Seo, Chang Hye

1994-01-01

To characterize the MR findings of hypoxic-ischemic brain injury and to assess the value of the MR imaging. SE T1-, T2-weighted, and IR brain MR images of 44 infants and children with the past history of perinatal hypoxic insults were reviewed. Abnormal brain MR findings of 8 patients with birth history of prematurity and 36 patients with birth history of full-term/posterm including 7 with severe anoxic insult history, were compared in regard to the location and the character of the lesions. MRI demonstrated the followings; (1)abnormal signal intensity lesions of subcortical and/or deep cerebral white matter, cortex, and deep gray matter, (2)atrophy of the cerebral white matter, cortex and corpus callosum, with/without ventriculomegaly, and (3)delay in myelination. Periventricular and deep white matter lesions were demonstrated in the prematurity, the deep white matter lesions and/ or subcortical white matter lesions in the term/post-term, and deep gray matter lesions in the 7 patients with severe anoxic insults history. MR imaging was useful in the diagnosis of the hypoxic-ischemic brain injury, and the white and gray matter lesions were correlated with the time of the injury and the severity of hypoxic insult

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

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

2015-04-01

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

Energy metabolism in human melanoma cells under hypoxic and acidic conditions in vitro.

Science.gov (United States)

Skøyum, R; Eide, K; Berg, K; Rofstad, E K

1997-01-01

The response to treatment and the malignant progression of tumours are influenced by the ability of the tumour cells to withstand severe energy deprivation during prolonged exposure to hypoxia at normal or low extracellular pH (pHe). The objective of the present work was to demonstrate intertumour heterogeneity under conditions of microenvironment-induced energy deprivation and to investigate whether the heterogeneity can be attributed to differences in the capacity of the tumour cells to generate energy in an oxygen-deficient microenvironment. Cultures of four human melanoma cell lines (BEX-c, COX-c, SAX-c, WIX-c) were exposed to hypoxia in vitro at pHe 7.4, 7.0 or 6.6 for times up to 31 h by using the steel-chamber method. High-performance liquid chromatography was used to assess adenylate energy charge as a function of exposure time. Cellular rates of glucose uptake and lactate release were determined by using standard enzymatic test kits. The adenylate energy charge decreased with time under hypoxia in all cell lines. The decrease was most pronounced shortly after the treatment had been initiated and then tapered off. BEX-c and SAX-c showed a significantly higher adenylate energy charge under hypoxic conditions than did COX-c and WIX-c whether the pHe was 7.4, 7.0 or 6.6, showing that tumours can differ in the ability to avoid energy deprivation during microenvironmental stress. There was no correlation between the adenylate energy charge and the rates of glucose uptake and lactate release. Intertumour heterogeneity in the ability to withstand energy deprivation in an oxygen-deficient microenvironment cannot therefore be attributed mainly to differences in the capacity of the tumour cells to generate energy by anaerobic metabolism. The data presented here suggest that the heterogeneity is rather caused by differences in the capacity of the tumour cells to reduce the rate of energy consumption when exposed to hypoxia.

Neuroprotection by hypoxic preconditioning involves upregulation of hypoxia-inducible factor-1 in a prenatal model of acute hypoxia.

Science.gov (United States)

Giusti, Sebastián; Fiszer de Plazas, Sara

2012-02-01

The molecular pathways underlying the neuroprotective effects of preconditioning are promising, potentially drugable targets to promote cell survival. However, these pathways are complex and are not yet fully understood. In this study we have established a paradigm of hypoxic preconditioning based on a chick embryo model of normobaric acute hypoxia previously developed by our group. With this model, we analyzed the role of hypoxia-inducible factor-1α (HIF-1α) stabilization during preconditioning in HIF-1 signaling after the hypoxic injury and in the development of a neuroprotective effect against the insult. To this end, we used a pharmacological approach, based on the in vivo administration of positive (Fe(2+), ascorbate) and negative (CoCl(2)) modulators of the activity of HIF-prolyl hydroxylases (PHDs), the main regulators of HIF-1. We have found that preconditioning has a reinforcing effect on HIF-1 accumulation during the subsequent hypoxic injury. In addition, we have also demonstrated that HIF-1 induction during hypoxic preconditioning is necessary to obtain an enhancement in HIF-1 accumulation and to develop a tolerance against a subsequent hypoxic injury. We provide in vivo evidence that administration of Fe(2+) and ascorbate modulates HIF accumulation, suggesting that PHDs might be targets for neuroprotection in the CNS. Copyright © 2011 Wiley Periodicals, Inc.

Dual responsive promoters to target therapeutic gene expression to radiation-resistant hypoxic tumor cells

International Nuclear Information System (INIS)

Chadderton, Naomi; Cowen, Rachel L.; Sheppard, Freda C.D.; Robinson, Suzanne; Greco, Olga; Scott, Simon D.; Stratford, Ian J.; Patterson, Adam V.; Williams, Kaye J.

2005-01-01

Purpose: Tumor hypoxia is unequivocally linked to poor radiotherapy outcome. This study aimed to identify enhancer sequences that respond maximally to a combination of radiation and hypoxia for use in genetic radiotherapy approaches. Methods and materials: The influence of radiation (5 Gy) and hypoxia (1% O 2 ) on reporter-gene expression driven by hypoxia (HRE) and radiation (Egr-1) responsive elements was evaluated in tumor cells grown as monolayers or multicellular spheroids. Hypoxia-inducible factor-1α (HIF-1α) and HIF-2α protein expression was monitored in parallel. Results: Of the sequences tested, an HRE from the phosphoglycerate kinase-1 gene (PGK-18[5+]) was maximally induced in response to hypoxia plus radiation in all 5 cell lines tested. The additional radiation treatment afforded a significant increase in the induction of PGK-18[5+] compared with hypoxia alone in 3 cell lines. HIF-1α/2α were induced by radiation but combined hypoxia/radiation treatment did not yield a further increase. The dual responsive nature of HREs was maintained when spheroids were irradiated after delivery of HRE constructs in a replication-deficient adenovirus. Conclusions: Hypoxia-responsive enhancer element sequences are dually responsive to combined radiation and hypoxic treatment. Their use in genetic radiotherapy in vivo could maximize expression in the most radio-resistant population at the time of radiation and also exploit microenvironmental changes after radiotherapy to yield additional switch-on

Apparent diffusion coefficient histogram analysis of neonatal hypoxic-ischemic encephalopathy

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Cauley, Keith A. [University of Massachusetts Medical School, Department of Radiology, Worcester, MA (United States); New York Presbyterian Hospital, Columbia University Medical Center, Department of Radiology, New York, NY (United States); Filippi, Christopher G. [New York Presbyterian Hospital, Columbia University Medical Center, Department of Radiology, New York, NY (United States)

2014-06-15

Diffusion-weighted imaging is a valuable tool in the assessment of the neonatal brain, and changes in diffusion are seen in normal development as well as in pathological states such as hypoxic-ischemic encephalopathy (HIE). Various methods of quantitative assessment of diffusion values have been reported. Global ischemic injury occurring during the time of rapid developmental changes in brain myelination can complicate the imaging diagnosis of neonatal HIE. To compare a quantitative method of histographic analysis of brain apparent coefficient (ADC) maps to the qualitative interpretation of routine brain MR imaging studies. We correlate changes in diffusion values with gestational age in radiographically normal neonates, and we investigate the sensitivity of the method as a quantitative measure of hypoxic-ischemic encephalopathy. We reviewed all brain MRI studies from the neonatal intensive care unit (NICU) at our university medical center over a 4-year period to identify cases that were radiographically normal (23 cases) and those with diffuse, global hypoxic-ischemic encephalopathy (12 cases). We histographically displayed ADC values of a single brain slice at the level of the basal ganglia and correlated peak (s-sD{sub av}) and lowest histogram values (s-sD{sub lowest}) with gestational age. Normative s-sD{sub av} values correlated significantly with gestational age and declined linearly through the neonatal period (r {sup 2} = 0.477, P < 0.01). Six of 12 cases of known HIE demonstrated significantly lower s-sD{sub av} and s-sD{sub lowest} ADC values than were reflected in the normative distribution; several cases of HIE fell within a 95% confidence interval for normative studies, and one case demonstrated higher-than-normal s-sD{sub av}. Single-slice histographic display of ADC values is a rapid and clinically feasible method of quantitative analysis of diffusion. In this study normative values derived from consecutive neonates without radiographic evidence of

Postnatal morphology of hematoencephalic barrier in hypoxic lesion

Directory of Open Access Journals (Sweden)

E. V. Kikhtenko

2012-12-01

Full Text Available In infants with perinatal hypoxic lesion of the central nervous system swelling and death of the endothelium, thickening of the capillary basement membranes, karyorrhexis and plasmorrhexis of astrocytes are observed. The severity and degree of pathological changes depends on the time of hypoxic exposure (antenatal or intrapartum period and the term of postnatal life.

Radiation sensitivity of Merkel cell carcinoma cell lines

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Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Radiation sensitivity of Merkell cell carcinoma cell lines

International Nuclear Information System (INIS)

Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

1995-01-01

Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

Satellite-based empirical models linking river plume dynamics with hypoxic area andvolume

Science.gov (United States)

Satellite-based empirical models explaining hypoxic area and volume variation were developed for the seasonally hypoxic (O2 < 2 mg L−1) northern Gulf of Mexico adjacent to the Mississippi River. Annual variations in midsummer hypoxic area and ...

The experimental investigation of fibrinolytic system under the influence of flocalin in conditions of acute hypoxic kidney injury

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A. I. Gozhenko

2017-08-01

Full Text Available In the experiments on rats subjected to acute hypoxic histochemical nephropathy, caused by sodium nitrite and 2,4-dinitrophenol, fibrinolytic activities of blood plasma, urine, renal cortex, medulla, and papilla after treatment with flocalin – the activator of KATP channels, were studied­. It was shown that in the conditions of acute kidney hypoxic injury flocalin administration resulted in the increase and essential restoration of fibrinolysis in blood plasma diminished under hypoxia, which was due to the growth of non-enzymatic fibrinolysis, whereas in urine and renal medulla the appreciable increase of enzymatic fibrinolytic activity took place. Moreover, the treatment of hypoxic nephropathy animals by flocalin resulted in the marked restoration of kidney ion regulatory and protein excretory functions that proves the positive influence of KATP channels activation on the one of the biochemical mechanisms of acute kidney injury as well as the protective effect of flocalin in relation to tubular cells of nephron. The obtained results testify to the beneficial effects of KATP channels activation in the conditions of acute hypoxic kidneys injury.