Chronic Hypoxic Condition Research Articles

Chronic hypoxia drives the occurrence of ferroptosis in liver of fat greening (Hexagrammos otakii) by activating HIF-1α and promoting iron production

BackgroundHypoxia caused by global climate change and human activities has become a growing concern eliciting serious effect and damages to aquatic animals. Hexagrammos otakii is usually a victim of hypoxia which caused by high density aquaculture and high nutrient input. The mechanism underlying ferroptosis regulation after hypoxia-stress in liver of H. otakii, however, remains elusive. MethodsFor a duration of 15 days, expose the H. otakii to low concentrations of dissolved oxygen (3.4 ± 0.2 mg/L). Detecting alterations in the H. otakii liver tissue by chemical staining, immunohistochemistry, and electron microscopy. The expression variations of relevant genes in the liver of the H. otakii were simultaneously detected using Western blot and qPCR. A correlation analysis was performed between HIF-1α and iron ion expression in the liver of H. otakii following hypoxic stress. ResultsIn this study, we conducted the whole ferroptosis integrated analysis of H. otakii under chronic hypoxic condition. Reactive oxygen species (ROS) are highly accumulated under the hypoxia treatment (Superoxide Dismutase, SOD; Catalase, CAT), and which results in a significantly enhanced of lipid peroxidation (Lipid Peroxidation, LPO; Malondialdehyde, MDA; Aminotransferase, AST; Alanine aminotransferase, ALT) in liver tissue. The HIF-1α signaling is activated to cope with the hypoxia stress through strategies including changing iron ion concentration (Fe3+ and TFR1) to breaking the oxidation balance (GSH and GSH-Px), and enhancing ferroptosis gene expression (GPX4). The expression of genes related to ferroptosis pathway (DMT1, FTH1, STEAP3, ACSL4, γ-GCS, SLC7A11) is significantly upregulated and associated to the expression of iron and HIF-1α. ConclusionsIt is demonstrated that the HIF-1α/Fe3+/ROS/GPX4 axis is involved in promoting ferroptosis in fat greening hepatocytes following hypoxia-stress. Ultimately, our findings unveil a process by which hypoxic stress strongly encourages ferroptosis by triggering HIF-1α and boosting iron synthesis.

Association of NRF2 with HIF-2α-induced cancer stem cell phenotypes in chronic hypoxic condition

The acquisition of the cancer stem cell (CSC) properties is often mediated by the surrounding microenvironment, and tumor hypoxia is considered an important factor for CSC phenotype development. High levels of NRF2 (Nuclear Factor Erythroid 2-Like 2; NFE2L2), a transcription factor that maintains cellular redox balance, have been associated with facilitated tumor growth and therapy resistance. In this study, we investigated the role of NRF2 in hypoxia-induced CSC phenotypes in colorectal cancer cells. Chronic hypoxia for 72 h resulted in CSC phenotypes, including elevation of krupple-like factor 4 (KLF4) and octamer-binding transcription factor 4 (OCT4), and an increase in cancer migration and spheroid growth with concomitant hypoxia-inducible factor 2α (HIF-2α) accumulation. All these chronic hypoxia-induced CSC properties were attenuated following HIF-2α-specific silencing. In this chronic hypoxia model, NRF2 inhibition by shRNA-based silencing or brusatol treatment blocked HIF-2α accumulation, which consequently resulted in decreased CSC marker expression and inhibition of CSC properties such as spheroid growth. In contrast, NRF2 overactivation by genetic or chemical approach enhanced the chronic hypoxia-induced HIF-2α accumulation and cancer migration. As a molecular mechanism of the NRF2-inhibition-mediated HIF-2α dysregulation, we demonstrated that miR-181a-2-3p, whose expression is elevated in NRF2-silenced cells, targeted the HIF-2α 3′UTR and subsequently suppressed the chronic hypoxia-induced HIF-2α and CSC phenotypes. The miR-181a-2-3p inhibitor treatment in NRF2-silenced cells could restore the levels of HIF-2α and CSC markers, and increased cancer migration and sphere formation under chronic hypoxia. In line with this, the miR-181a-2-3p inhibitor transfection could increase tumorigenicity of NRF2-silenced colorectal cancer cells. Collectively, our study suggests the involvement of NRF2/miR181a-2-3p signaling in the development of HIF-2α-mediated CSC phenotypes in sustained hypoxic environments.

Silibinin efficacy in a rat model of pulmonary arterial hypertension using monocrotaline and chronic hypoxia

BackgroundC-X-C chemokine receptor type 4 (CXCR4) may be involved in the development of pulmonary arterial hypertension (PAH). CXCR4 inhibitor AMD3100 was described to have a positive effect on the prevention of pulmonary arterial muscularization in PAH models. Silibinin is a traditional medicine that has an antagonistic effect on CXCR4. We investigated the effect of silibinin using rat models of PAH.MethodsPAH was induced by a single subcutaneous injection of monocrotaline. The rats were maintained in a chronic hypoxic condition (10% O2) with or without silibinin. To evaluate the efficacy of silibinin on PAH, right ventricular systolic pressure (RVSP), Fulton index (weight ratio of right ventricle to the left ventricle and septum), percent medial wall thickness (% MT), and vascular occlusion score (VOS) were measured and calculated. Immunohistochemical analysis was performed targeting CXCR4 and c-Kit. Reverse transcription-quantitative polymerase chain reaction was performed for the stem cell markers CXCR4, stromal cell derived factor-1 (SDF-1), c-Kit, and stem cell factor (SCF), and the inflammatory markers monocyte chemoattractant protein 1 (MCP1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα). Statistical analyses were performed using t-test and one-way analysis of variance with Bonferroni’s post hoc test.ResultsSilibinin treatment for 1 week reduced RVSP and Fulton index. Treatment for 2 weeks reduced RVSP, Fulton index, % MT, and VOS, as well as downregulating the expression of CXCR4, SDF-1, and TNFα in pulmonary arteries. In contrast, treatment for 3 weeks failed to ameliorate PAH. The time-course study demonstrated that RVSP, Fulton index, % MT, and VOS gradually increased over time, with a decrease in the expression of CXCR4 and TNFα occurring after 2 weeks of PAH development. After 3 weeks, SDF-1, c-Kit, and SCF began to decrease and, after 5 weeks, MCP1 and IL-6 gradually accumulated.ConclusionsThe CXCR4 inhibitor silibinin can ameliorate PAH, possibly through the suppression of the CXCR4/SDF-1 axis, until the point where PAH becomes a severe and irreversible condition. Silibinin results in reduced pulmonary arterial pressure and delays pulmonary arteriolar occlusion and pulmonary vascular remodeling.

High Altitude Pulmonary Hypertension

The effects of hypoxia on the human organism has been considered doubly fascinating by the scientific community. The knowledge of the discrete mechanisms allowing the acclimatization both at genetic level or through the cell mediators production in addition to the macroscopic responses of the cardio-circulatory and ventilatory systems to a hypoxic environment has been progressively developed since the last century; moreover granting a safer stay in hypoxic conditions not only for the residents but also for the different cathegories of workers, sportsmen and tourists has been considered a worthy aim of the medical activity. The effects of hypoxia were simulated in laboratory by means of an induced low pressure environment (normobaric hypoxia) or tested on the subjects at different levels of altitude (hypobaric hypoxia). Far from describing all the physiological and pathological responses of the organism, in this review, the authors expose the state of the art in the knowledge of the responsiveness of the pulmonary circle to the acute or chronic hypoxic condition, its possible progression to the pulmonary arterial hypertension, the latter being more appropriately named High-Altitude Pulmonary Hypertension. The currently available therapeutic options in the treatment of High-Altitude Pulmonary Hypertension are also reviewed.

Chronic obstructive pulmonary disease-induced autonomic dysfunction may be associated with cerebral blood flow regulation and brain function.

Chronic obstructive pulmonary disease (COPD) increases the risk of cerebrovascular disease and brain dysfunction, such as ischaemic stroke and dementia, in proportion to its severity. Cerebral blood flow (CBF) regulation may be a key factor for this phenomenon, but the physiological mechanisms of COPD pathogenesis regarding cerebrovascular disease are yet to be established. Physiologically, COPD is a chronic hypoxic condition, and long-term oxygen therapy improves survival in patients with COPD and severe resting hypoxaemia. Given this background, Hoiland et al. (2018) have reported, in this issue of Experimental Physiology, the effects of oxygen therapy on cerebral oxygen delivery and neurovascular function in patients with COPD. The authors demonstrated that oxygen therapy improved cerebral oxygen delivery by increasing peripheral oxyhaemoglobin saturation () and enhanced neurovascular function in patients with COPD. These findings suggested that such oxygen delivery and functional neurovascular improvements may provide a physiological link between oxygen therapy and the reduced risk of cerebrovascular disease in patients with COPD. Thus, oxygen therapy appears beneficial for patients with COPD. On the contrary, oxygen therapy did not alter volumetric global CBF (Hoiland et al., 2018), suggesting that the cerebral vasculature has a low sensitivity to the acute normalization of . From these results, it can be suspected that the contribution of CBF regulation to COPD-induced cerebrovascular disease or brain dysfunction is likely to be minimal. In patients with COPD receiving oxygen therapy, we should consider cerebral oxygen delivery and cerebral haemodynamics and other physiological mechanisms potentially induced by hypoxaemia to understand the physiological mechanisms of this positive phenomenon. One example includes autonomic dysfunction, which is commonly observed in patients with COPD. Indeed, a previous study (van Gestel, Kohler, & Clarenbach, 2012) has demonstrated that autonomic dysfunction through sympathetic nervous system overactivity might contribute to the initiation and progression of cardiovascular disease in the examined population. However, the contributions of autonomic dysfunction to COPD-induced cerebrovascular disease and brain dysfunction may not be simple, because autonomic dysfunction modifies cerebral vasculature regulation (cerebral autoregulation and cerebral CO2 reactivity), the arterial baroreflex and the respiratory system (Ogoh & Ainslie, 2009). Indeed, baroreflex function is attenuated in patients with COPD. Importantly, the baroreflex simultaneously influences the respiratory system (ventilation and tidal volume) and the control of arterial pressure (Brunner, Sussman, Greene, Kallman, & Shoukas, 1982). Moreover, there is an interaction between cerebral vasculature regulation and the arterial baroreflex or respiratory system (Ogoh & Ainslie, 2009). Taken together, we cannot rule out the possibility that autonomic dysfunction might affect cerebral vasculature and function in the examined population both directly and indirectly. Indeed, another study (Bartels, Gonzalez, Kim, & De Meersman, 2000) demonstrated that oxygen therapy increased baroreflex sensitivity in patients with COPD, indicating that oxygen supplementation in this population significantly and favourably altered autonomic activity. Thus, oxygen therapy-modified autonomic function may be associated with improved survival in patients with COPD. Regarding this concept, the duration or severity of COPD may cause different functional autonomic adaptations and may result in different effects of oxygen therapy on the cerebral vasculature. The work of Hoiland et al. (2018) has clearly described the mechanisms through which oxygen therapy improved survival in patients with COPD. However, in order to identify the mechanisms underlying these effects, we should conduct additional, detailed investigations of the physiological regulatory mechanisms associated with various autonomic functions.

Expression and specific activities of carbamoyl phosphate synthetase 1 in chronic hypoxic rats

Background: Urea biosynthesis is a very important process in the liver which needs ATP, CO2 and functional mitochondria or aerobic condition. Liver can adapt to hypoxic condition, generally and locally. This study aimed to analyze the effect of chronic hypoxia on liver urea biosynthesis as indicated by the level and specific activity of mRNA of carbamoyl phosphate synthetase 1 (CPS1), a key enzyme in urea biosynthesis in hypoxic rats.Methods: 20 male Sprague-Dawley rats were placed in hypoxic chamber supplied by a mixture of 10% O2 and 90% N2. Five rats were sacrificed at 1, 3, 5, and 7 days after exposure. Liver homogenates were analyzed for HIF-1 (hypoxia inducible factor-1) by ELISA, CPS1 mRNA by real time RT-PCR and CPS1 enzymatic specific activities by Pierson method. Data were analyzed by ANOVA test and Pearson correlation.Results: The HIF-1 in liver increased significantly, as well as CPS1 mRNA and CPS1 enzymatic activities (p<0.05). There was a strong correlation (r=0.618; p<0.01) between the level of CPS1 mRNA and CPS1 enzymatic activities, moderate correlation between HIF-1 and CPS1 mRNA (r=0.419; p<0.05) but no correlation between HIF-1 and CPS1 enzymatic activities. The study indicated that urea biosynthesis in liver was affected by hypoxia and partially under HIF-1 regulation. The study also found increase of urea and NH3 biosynthesis related to proteolysis as indicated by the decrease of total body weight and liver weight.Conclusion: There was an increase in the expression and specific activities of CPS1 in urea biosynthesis as a result of increasing proteolysis in chronic hypoxic condition.

H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl) secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA). This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2), and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159), and drastically lowered from the third to seventh day (0.289; 0.108; 0.062). Specific activities of H,K-ATPase was slightly higher in the first day (0.765), then was lowered in the third (0.685) and fifth day (0.655), and was higher in the seventh day (0.884). The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343). Specific activities of total CA was low in the first day (0.083), and was higher from the third to seventh day (0.111; 0.136; 0.144).Conclusion: In hypoxia condition, expression of H,K-ATPase and CA9 mRNA were decreased, but the specific activity of H,K-ATPase and total CA were increased.

Placental histology in clinically unexpected severe fetal acidemia at term

BackgroundFetal acidemia at birth is defined as a newborn condition wherein the cord blood pH value is less than 7.0. It could represent an association with newborn brain damage; therefore, it is important to investigate which conditions precipitate its occurrence. No extensive placental analysis has been performed in cases of acidotic newborns delivered from low-risk pregnancies. AimsTo study placental characteristics in cases with severe fetal acidemia. Study designRetrospective case–control study. Subject34 cases, 102 controls. Outcome measuresUmbilical artery pH was measured at delivery from a doubly clamped portion of the cord. Placental characteristics were compared between cases with severe fetal acidemia (cord pH at birth <7.0) and controls (normal pH at birth) in term low-risk pregnancies. ResultsMacroscopic placental and umbilical cord characteristics were comparable in cases and controls whereas histological characteristics exhibited differences: diffuse villous edema, increased number of syncytial knots and villous branching abnormalities significantly affected cases more frequently than controls. Diffuse villous edema is related to fetal vascularization and associated with an increase of venous pressure; in our low-risk population, it is conceivable that these changes of fetal flow and pressure occurred in labor during the alteration of fetal heart rate. An increased number of syncytial knots and villous branching abnormalities have been previously associated with chronic placental hypoxic condition; in our low-risk population they could reflect a clinically undetectable hypoxic situation that acted during pregnancy reducing fetal resources to bear labor and delivery. ConclusionsPlacental histology provides useful information related to fetal acidemia in low-risk term pregnancy.

Novel poly (ADP-ribose) polymerase inhibitor, AZD2281, enhances radiosensitivity of both normoxic and hypoxic esophageal squamous cancer cells.

Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma (ESCC). However, the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells, particularly those under hypoxic condition, exhibit radioresistance. The aim of this study was to determine whether or not AZD2281, a potent poly (ADP-ribose) polymerase (PARP) inhibitor, could enhance the radiation sensitivity of two ESCC cell lines, namely ECA109 and TE13. The radiosensitizing effect of AZD2281 was evaluated on the basis of cell death, clonogenic survival and tumor xenograft progression. AZD2281 alone was slightly toxic to ESCC cell lines. Apoptosis was increased and clonogenic survival was decreased in both cell lines when AZD2281 was combined with ionizing radiation (IR) under normoxic condition. AZD2281 enhanced IR-induced apoptosis to a more significant level under chronic hypoxic condition (0.2% O(2), 48 hour) than under normoxic condition. AZD2281 also slightly enhanced clonogenic cell death under chronic hypoxic condition compared with that under normoxic condition. This result could be associated with increased radiation-induced DNA double-strand breaks (DSB), decreased DSB repair and increased apoptosis of ESCC cells. Furthermore, homologous recombination (HR) protein Rad51 expression and focus formation were decreased in ESCC cells exposed to moderate chronic hypoxic condition (0.2% O(2), 48 hour); this result indicated that chronic hypoxic ESCC cells were HR deficient, possibly causing contextual synthetic lethality with PARP inhibitor in radiation sensitization. AZD2281 was also a radiation sensitizer in ESCC tumor xenograft models. Hence, in vitro and in vivo findings provide evidence that AZD2281 potently sensitizes ESCC cells to X-ray irradiation. The selective cell killing of HR-defective hypoxic cells contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition.

Abstract 1651: Targeting Vav3 oncogene enhances docetaxel-induced apoptosis in LNCaP cells under chronic hypoxia

Abstract Introduction and Objective: We previously reported that chronic hypoxic condition (1% oxygen for over six months) induced Vav3 oncogene expression with androgen-independent growth and malignant behavior in LNCaP cells. And overexpression of Vav3 oncogene promoted the growth of LNCaP cells with increased invasion and migration under normoxic condition. The objective of this study was to examine the effect of Vav3 siRNA on cell proliferation and apoptosis in docetaxel-treated LNCaP cells under chronic hypoxic condition. Methods: We analyzed the sensitivity of docetaxel in LNCaP cells under normoxic and chronic hypoxic conditions by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) and flow cytometric analyses. The effects of Vav3 siRNA on cell growth and apoptosis were examined under chronic hypoxia with or without docetaxel treatment. To seek a molecular understanding of Vav3 siRNA induced apoptosis, we analyzed alterations in the growth signaling components in the PI3K/Akt and ERK mediated pathways by immunoblot analysis. Results: Overexpression of Vav3 was observed in LNCaP cells under chronic hypoxia compared with its expression under normoxia. Although the growth rates were reduced in a dose- and time-dependent manner by docetaxel in LNCaP cells under both normoxic and chronic hypoxic conditions, these effects were significantly decreased in LNCaP cells under chronic hypoxia. In addition, we found that docetaxel induced apoptosis following activation of caspase-3 and cleaved poly (ADP-ribose) polymerase, and that the interrupting the Vav3 signaling pathway using siRNA enhanced docetaxel sensitivity of LNCaP cells under chronic hypoxia and led to apoptosis. A profound activation of PI3K/Akt pathway was observed under chronic hypoxic condition. Treatment with Vav3 siRNA showed weak inhibition of Akt phosphorylation and treatment with Vav3 siRNA plus docetaxel strongly inhibited this signaling. Conclusions: Our results showed that Vav3 siRNA enhances docetaxel-induced apoptosis in LNCaP cells under chronic hypoxia and that targeting Vav3 oncogene may have important practical implications for managing hormone refractory prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1651. doi:10.1158/1538-7445.AM2011-1651

418 TARGETING VAV3 ONCOGENE ENHANCES DOCETAXEL SENSITIVITY VIA INDUCTION OF APOPTOSIS IN LNCAP CELLS UNDER CHRONIC HYPOXIA

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011418 TARGETING VAV3 ONCOGENE ENHANCES DOCETAXEL SENSITIVITY VIA INDUCTION OF APOPTOSIS IN LNCAP CELLS UNDER CHRONIC HYPOXIA Takeo Nomura, Kenichi Hirai, Mutsushi Yamasaki, Fuminori Sato, and Hiromitsu Mimata Takeo NomuraTakeo Nomura Oita, Japan More articles by this author , Kenichi HiraiKenichi Hirai Oita, Japan More articles by this author , Mutsushi YamasakiMutsushi Yamasaki Oita, Japan More articles by this author , Fuminori SatoFuminori Sato Oita, Japan More articles by this author , and Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.507AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The Vav family are Rho/Rac guanosine nucleotide exchange factor (GEFs) consisting of three members in mammalian cells and Vav3 enhances AR activity during progression to androgen independence. We previously reported that chronic hypoxic condition (1% oxygen for over six months) induced Vav3 oncogene expression with androgen-independent growth and malignant behavior in LNCaP cells. And overexpression of Vav3 oncogene promoted the growth of LNCaP cells with increased invasion and migration under normoxic condition. The objective of this study was to examine the effect of Vav3 siRNA on cell proliferation and apoptosis in docetaxel-treated LNCaP cells under chronic hypoxic condition. METHODS We analyzed the sensitivity of docetaxel in LNCaP cells under normoxic and chronic hypoxic conditions by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) analysis. The effects of Vav3 siRNA on cell growth and apoptosis were examined under chronic hypoxia with or without docetaxel treatment by WST-1 and flow cytometric analyses. To seek a molecular understanding of Vav3 siRNA induced apoptosis, we analyzed alterations in the growth signaling components in the PI3K/Akt and ERK mediated pathways and caspase activation by immunoblot analysis. RESULTS Overexpression of Vav3 was observed in LNCaP cells under chronic hypoxia compared with its expression under normoxia. Although the growth rates were reduced in a dose- and time-dependent manner by docetaxel in LNCaP cells under both normoxic and chronic hypoxic conditions, these effects were significantly decreased in LNCaP cells under chronic hypoxia. In addition, we found that docetaxel induced apoptosis following activation of caspase-3, -9 and cleaved poly (ADP-ribose) polymerase, and that the interrupting the Vav3 signaling pathway using siRNA enhanced docetaxel sensitivity of LNCaP cells under chronic hypoxia and led to apoptosis. Although a profound activation of PI3K/Akt pathway was observed under chronic hypoxic condition, treatment with Vav3 siRNA showed weak inhibition of Akt phosphorylation. CONCLUSIONS Our results showed that Vav3 siRNA enhances docetaxel-induced apoptosis in LNCaP cells under chronic hypoxia via activation of caspase cascade and that targeting Vav3 oncogene may have important practical implications for managing castration resistant prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e168 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeo Nomura Oita, Japan More articles by this author Kenichi Hirai Oita, Japan More articles by this author Mutsushi Yamasaki Oita, Japan More articles by this author Fuminori Sato Oita, Japan More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

405 VAV3, A POSSIBLE KEY MOLECULE, ENHANCES ANDROGEN-INDEPENDENT GROWTH AND THE MALIGNANT POTENTIAL OF PROSTATE CANCER CELLS UNDER CHRONIC HYPOXIA

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011405 VAV3, A POSSIBLE KEY MOLECULE, ENHANCES ANDROGEN-INDEPENDENT GROWTH AND THE MALIGNANT POTENTIAL OF PROSTATE CANCER CELLS UNDER CHRONIC HYPOXIA Kenichi Hirai, Takeo Nomura, Mutsushi Yamasaki, Takanori Matsubara, Fuminori Sato, Masatsugu Moriyama, and Hiromitsu Mimata Kenichi HiraiKenichi Hirai Oita, Japan More articles by this author , Takeo NomuraTakeo Nomura Oita, Japan More articles by this author , Mutsushi YamasakiMutsushi Yamasaki Oita, Japan More articles by this author , Takanori MatsubaraTakanori Matsubara Oita, Japan More articles by this author , Fuminori SatoFuminori Sato Oita, Japan More articles by this author , Masatsugu MoriyamaMasatsugu Moriyama Oita, Japan More articles by this author , and Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.494AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hypoxia occurs in many solid tumors, including prostate cancer, and enhances tumor progression and treatment resistance. Our previous studies showed that chronic hypoxia promoted androgen-independent growth and malignant cell behavior in a human prostate cancer cell line. From the results of multi-pathway analysis of gene expression of LNCaP cells under a chronic hypoxic condition, we specifically focused on the biological functions of Vav3 gene in vitro. METHODS We analyzed the gene expression profiles of LNCaP cells under normoxic, acute hypoxic (1% oxygen for forty-eight hours), and chronic hypoxic conditions (cultured over six months). Comprehensive mRNA profiling was performed using an Agilent 44K array platform, and we investigated the association between elevated genes and tumor behavior by the applications of Ingenuity Pathways Analysis. From the results, we identified the Vav3 gene and closely analyzed the details of the gene in vitro. We validated the expression by quantitative RT-PCR and Western blot analyses. To analyze the function of Vav3, knockdown by siRNA was performed. Next, we transfected LNCaP with the Vav3 cDNA expression vector (control cells were transfected with neo expression vector) and isolated stable clones with high or low expression. The growth of cancer cells was evaluated in vitro. Also, we examined cell invasiveness and migration using the Matrigel invasion assay. For cell cycle analysis, flow cytometric analysis of propidium iodide-stained nuclei was carried out. RESULTS The results were as follows: (1) Vav3 mRNA and protein expression levels were increased in LNCaP cells under chronic hypoxia, respectively; (2) chronic hypoxia increased both the invasiveness and migration of LNCaP cells; (3) LNCaP cells under chronic hypoxia showed less invasiveness and cell motility as well as suppressed their growth rates by interrupting Vav3 expression using Vav3 siRNA; (4) Vav3 expression positively correlated with the growth, invasiveness, and motility of LNCaP cells; and (5) interrupting the Vav3 signaling axis with siRNA led to the apoptosis of LNCaP cells via the mitochondrial pathway, with increased activation of caspases-3, and -9 and cleaved PARP. CONCLUSIONS Our results showed that chronic hypoxia promoted the malignant potential of prostate cancer cells via the overexpression of Vav3. Therefore, targeting the Vav3 signaling pathway could prove to be a promising approach for the treatment of progressive or castration resistant prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e163-e164 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenichi Hirai Oita, Japan More articles by this author Takeo Nomura Oita, Japan More articles by this author Mutsushi Yamasaki Oita, Japan More articles by this author Takanori Matsubara Oita, Japan More articles by this author Fuminori Sato Oita, Japan More articles by this author Masatsugu Moriyama Oita, Japan More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The effect of hypoxia mimetic cobalt chloride on the expression of EC-SOD in 3T3-L1 adipocytes

It is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectin and tumor necrosis factor-α (TNF-α). Recently, it has been reported that adipose tissue can suffer a chronic hypoxic condition during hypertrophy of adipocytes, and this condition leads to the dysregulation of adipocytokines. Further, hypoxic adipocytes are in an increased oxidative stress. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from reactive oxygen species (ROS) by scavenging superoxide anion. Previous reports showed that plasma EC-SOD levels in type 2 diabetes patients were significantly and inversely related to the body mass index, homeostasis model assessment-insulin resistance index; however, the mechanisms of EC-SOD and adiponectin reductions during hypoxia remain poorly understood. Here, we demonstrate that cobalt chloride (CoCl2), a hypoxia mimetic, decreases EC-SOD and adiponectin in 3T3-L1 adipocytes by intracellular ROS-independent, but TNF-α and c-jun N-terminal kinase (JNK)-dependent mechanisms. From these results, it is possible that TNF-α is a key regulator of the reduction of EC-SOD and adiponectin in CoCl2-treated 3T3-L1 adipocytes, and we speculated that the reduction of EC-SOD and adiponectin would lead to and/or promote metabolic disorders.

Abstract 442: Down-regulation of MET/RON receptor tyrosine kinases in colon cancer cells under chronic hypoxia as a mechanism for resistance towards targeted therapy

Abstract Hypoxia is a pathogenic factor observed in solid tumors, which plays a critical role in tumor malignancy and drug resistance. The MET and RON receptor tyrosine kinases belong to a unique proto-oncogene family and contribute to tumorigenic progression and malignant phenotypes in various epithelial cancers. To address their pathogenic significance under chronic hypoxic condition (1% O2, 5% CO2 for 5 days), functional analysis of MET and RON were conducted using colon cancer HCT116 and HT29 cells as models. Contrary to previous reports, we consistently observed that levels of MET and RON were significantly decreased over a period of 5 days under chronic hypoxic condition as documented by Western blot and cell-surface fluorescent analysis. The reduction was also accompanied with decreased in both spontaneous and ligand (HGF/MSP)-induced tyrosine phosphorylation manners, which affected the downstream signaling cascades. Moreover, we observed that hypoxia-induced HIF-1α expression (known as a positive regulator for both receptors) is unable to sustain MET or RON expression, suggesting that the receptor down-regulation is mediated through a HIF-1α-independent mechanism. RT-PCR analysis revealed that diminished MET and RON expression occurs at the mRNA transcription level. However, increase in protein degradation also contributed to the observed reduction. Functional studies further demonstrated that chronic hypoxia facilitates colon cancer cells to develop a unique pathogenic phenotype. This phenotype was featured by reduced cell migration, decreased cell proliferation, and lack of S phase in cell cycle profile. Tumor cells were also insensitive in response towards HGF or MSP-induced cell movement and replication. These cancer cells were resistant to cytotoxic effects induced by MET-RON duel small molecule inhibitors such as PHA665752 and compound I. As shown by cytotoxicity assay, the IC50 values of these inhibitors in cell survival were significantly increased in cancer cells under chronic hypoxic treatment compared to normoxic cells. In conclusion, our results demonstrate that under chronic hypoxia colon cancer cells developed a pathogenic phenotype characterized by diminished expression of MET and RON. Such a change contributes to the insensitivity of tumor cells towards cytotoxic activities of specifically targeted drugs. Thus, diminished expression of MET and RON under chronic hypoxia is a potential mechanism that affects the effectiveness of targeted therapy in the treatment colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 442.

Effects of ginkgo biloba extract EGb761 on expression of RAGE and LRP-1 in cerebral microvascular endothelial cells under chronic hypoxia and hypoglycemia

Alzheimer's disease (AD), characterized by accumulation of amyloid-beta protein (Abeta) in brain parenchyma, is closely associated with brain ischemia. Decreased clearance of Abeta from brain is the main cause of Abeta accumulation in sporadic AD. However, the mechanisms underlying ischemia-mediated AD pathogenesis remain unclear. The receptor for advanced end glycation products (RAGE) and low-density lipoprotein receptor-related protein-1 (LRP-1) expressed at blood brain barrier (BBB) are actively involved in Abeta clearance. RAGE is thought to be a primary transporter of Abeta across BBB into the brain from the systemic circulation, while LRP-1 mediates the transport of Abeta out of the brain. Ginkgo biloba extract EGb761, a traditional Chinese medicine, has been widely used in the treatment of AD. To investigate the effects of EGb761 on the expression of RAGE and LRP-1 in endothelial cells in response to ischemic injury, we cultured bEnd.3 cells, an immortalized mouse cerebral microvessel endothelial cell line, under a chronic hypoxic and hypoglycemic condition (CHH) to mimic ischemic injury of BBB, and then treated with EGb 761. We found that EGb 761 markedly ameliorated the damage (evaluated by MTT assay) from CHH. Moreover, we demonstrated that CHH led to a significant increase in the expression of RAGE both at the mRNA and protein levels at all times (24, 36, and 48 h), conversely; CHH induced a dramatic decrease in LRP-1 mRNA and protein expression at both 36 and 48 h. The results indicated that CHH has differential effects on the expression of RAGE and LRP-1. Furthermore, EGb761 significantly reversed CHH-induced upregulation of RAGE expression and downregulation of LRP-1 expression. Our findings suggest that EGb761 favor clearance of Abeta via regulating the expression of RAGE and LRP-1 during brain ischemia. This may provide a new insight into a possible molecular mechanism underlying brain ischemia-mediated AD pathogenesis, and potential therapeutic application of EGb 761 in treatment of AD.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective To investigate the effects of calcium-activated chloride (ClCa) channels on proliferation of pulmonary artery smooth muscle cells(PASMCs) in rats under chronic hypoxic condition. Methods The cultured PASMCs were placed under normoxic and chronic hypoxic conditions:The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca 2+ concentration ([Ca 2+] i) in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope: In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi's bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S+G 2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from (28.6 ± 1.0)% to (16.0 ± 1.6)% and the number of G 0G 1 PASMCs significantly increased from (71.4 ± 1.9)% to (83.9 ± 1.6)% ( P < 0.01). In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from (81 ± 6)% to (27 ± 7)%( P < 0.01). The expression of c-fos and c-jun were significantly increased in chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ± 0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively ( P < 0.01); Under chronic hypoxic conditions, [Ca 2+] i was increased; The NFA and IAA-94 decreased it from (281.8 ± 16.5)nmol/L to (117.7±15.4)nmol/L( P < 0.01). Conclusion Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

Cobalt chloride decreases EC-SOD expression through intracellular ROS generation and p38-MAPK pathways in COS7 cells

It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl2)-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl2.

Relationship of intracellular free Ca2+ concentration and calcium-activated chloride channels of pulmonary artery smooth muscle cells in rats under hypoxic conditions

To investigate the relationship between intracellular free Ca2+ concentration ([Ca2+]i) and calcium-activated chloride (Clca) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca2+ indicator Fura-2/AM was used to observe [Ca2+]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay. The Clca channel blockers niflumic acid (NFA) and indanyloxyacetic acid (IAA-94) exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca2+]i was increased. Under normoxic condition, [Ca2+]i was (123.63 +/- 18.98) nmol/L, and in hypoxic condition, [Ca2+]i was (281.75 +/- 16.48) nmol/L (P<0.01). Under normoxic condition, [Ca2+]i showed no significant change and no effect on Clca channels was observed (P> 0.05). Chronic hypoxia increased [Ca2+]i which opened Clca channels. The NFA and IAA-94 blocked the channels and decreased [Ca2+]i from (281.75 +/- 16.48) nmol/L to (117.66 +/- 15.36) nmol/L (P<0.01). MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency (A value) from 0.459 +/- 0.058 to 0.224 +/- 0.025 (P<0.01). Hypoxia increased [Ca2+]i which opened Clca channels and had a positive-feedback in [Ca2+]i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Clca channel may play a part in the regulation of proliferation of PASMCs.

NEUROGLOBIN, A NEW OXYGEN BINDING PROTEIN IS PRESENT IN THE CAROTID BODY AND INCREASES AFTER CHRONIC INTERMITTENT HYPOXIA.

Neuroglobin (Ngb) is a member of a gas binging heme-proteins present in the vertebrate brain, which enhances O2 supply to neural components. It has been suggested that Ngb may contribute to neuronal survival, because the level of Ngb is augmented during ischemia and hypoxia. Since the CB is the main oxygen chemoreceptor in the arterial blood, we tested if Ngb is present in the CB of normoxic rats and if chronic hypoxic condition may increase its level. Two groups of six male Wistar rats weighting 200–250 g were used. One group kept at room air (21% O2) was used as a control, while the other group was subjected to alternative cycles of 12 hours of hypoxia (10% O2) and normoxia (21% O2) during 12 days in a Plexiglas chamber. Rats were anaesthetized with sodium pentobarbital (40 mg/kg) and the CBs were dissected. The presence of Ngb in the CB tissue was detected by immunohistochemistry using a polyclonal antibody (E-16, Santa Cruz Biotech Antibodies). The immunihistochemical staining was analyzed using and histochemistry software package (Image-proplus 4.5) for the densitometric analysis. We found that Ngb is present in the normoxic CB, presumable in the glomus cells. The immunoreactivity increased significantly in the chronic intermittent hypoxic group. In fact, the integrated optical intensity of Ngb increased from 29.3 ± 6.7% in the control to 61.7± 8.6% in the hypoxic group. It is known that Ngb may act as NO scavenger and participate in the detoxication of reactive oxygen species, which are presumable, generated under hypoxia. We cannot not excluded that Ngb may act as an O2 sensor.

The expression of a novel stress protein ‘150-kDa oxygen regulated protein’ in sudden infant death

The oxygen regulated protein 150-kDa (ORP-150) is only induced in hypoxic conditions. We performed an immunohistochemical and morphometrical study on the expression of ORP-150 in the brains of sudden infant death (SID) victims. The cerebral cortexes of 18 infants were used for this study. Each tissue section was incubated with anti-ORP-150 polyclonal antibodies and the number of ORP-150 positive cells was counted. In the cluster analysis, the 18 cases were classified into three groups (A–C groups). Group A was composed of six sudden infant death syndrome (SIDS) cases and its mean value of ORP-150 positive cells was 66.75±3.44, Group B (six severe respiratory infectious disease such as pneumonia and bronchitis including sepsis): 39.50±2.52 and Group C (five SIDS and one severe respiratory infectious disease): 16.00±2.92, respectively. These results might reflect chronic hypoxic condition before death, because ORP-150 is only induced when a hypoxic condition exist, but not acute hypoxia. And chronic hypoxic state is likely to be antecedent to SIDS. Therefore, immunohistochemical analysis of OPR-150 in the brain of SID cases may be very useful to differentiate between SIDS and acute asphyxia.