Hypoxia-induced Lung Research Articles

My Journey in Academia as a Lipid Biochemist.

An overview of Prof. Viswanathan Natarajan’s journey in academia as a mentor, teacher, and lipid scientist for nearly 50 years is presented. As a graduate student, Dr. Natarajan interrogated biosynthesis and catabolism of phospholipids in the developing brain; however, in the last five decades, he has been investigating the role of sphingolipids and sphingolipid-metabolizing enzymes in pulmonary endothelial cells, epithelial cells, and fibroblasts under normal conditions and during various lung pathologies such as sepsis, asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, bronchopulmonary dysplasia, and lung cancer. His recent work on sphingosine-1-phosphate and lysophosphatidic acid metabolism in pre-clinical animal models has identified small molecule inhibitors in the signaling pathways that could have therapeutic potential in ameliorating pulmonary fibrosis, hypoxia-induced pulmonary hypertension, lung cancer, and bronchopulmonary dysplasia. Future research in bioactive lipids in combination with OMICS should unravel the importance of various lipid mediators as modulators of cell function under normal and pathological conditions.

An Oxygen-Concentration-Controllable Multiorgan Microfluidic Platform for Studying Hypoxia-Induced Lung Cancer-Liver Metastasis and Screening Drugs.

Various cancer metastasis models based on organ-on-a-chip platforms have been established to study molecular mechanisms and screen drugs. However, current platforms can neither reveal hypoxia-induced cancer metastasis mechanisms nor allow drug screening under a hypoxia environment on a multiorgan level. We have developed a three-dimensional-culture multiorgan microfluidic (3D-CMOM) platform in which the dissolved oxygen concentration can be precisely controlled. An organ-level lung cancer and liver linkage model was established under normoxic/hypoxic conditions. A transcriptomics analysis of the hypoxia-induced lung cancer cells (A549 cells) on the platform indicated that the hypoxia-inducible factor 1α (HIF-1α) pathway could elevate epithelial-mesenchymal transition (EMT) transcription factors (Snail 1 and Snail 2), which could promote cancer metastasis. Then, protein detection demonstrated that HIF-1α and EMT transcription factor expression levels were positively correlated with the secretion of cancer metastasis damage factors alpha-fetoprotein (AFP), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (γ-GT) from liver cells. Furthermore, the cancer treatment effects of HIF-1α inhibitors (tirapazamine, SYP-5, and IDF-11774) were evaluated using the platform. The treatment effect of SYP-5 was enhanced under the hypoxic conditions with fewer side effects, similar to the findings of TPZ. We can envision its wide application in future investigations of cancer metastasis and screening of drugs under hypoxic conditions with the potential to replace animal experiments.

TOPK mediates hypoxia-induced epithelial-mesenchymal transition and the invasion of nonsmall-cell lung cancer cells via the HIF-1α/snail axis

Hypoxia has been suggested to induce epithelial-mesenchymal transition (EMT) in various cancer types via the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). Here, we demonstrated that TOPK upregulates EMT and the invasion of H460 nonsmall-cell lung cancer cells through the induction of the HIF-1α/Snail axis and hypoxic signaling. The expression of endogenous TOPK, phosphorylated TOPK, HIF-1α and Snail was significantly increased upon hypoxia exposure, but TOPK depletion markedly abrogated the induced mRNA and protein levels of HIF-1α and Snail. Interestingly, TOPK knockdown restored the hypoxia-induced suppression of E-cadherin and diminished hypoxia-induced N-cadherin expression. In addition, Snail depletion suppressed hypoxia-induced N-cadherin expression, which was attenuated by TOPK knockdown. Moreover, knockdown of Snail decreased hypoxia-induced nonsmall-cell lung cancer cell migration and invasion, which were suppressed by TOPK depletion. In summary, we conclude that TOPK positively regulates HIF-1α expression through hypoxia signaling and thereby promotes Snail expression, leading to EMT and the invasion of nonsmall-cell lung cancer cells. These findings suggest that TOPK plays a critical role as a novel mediator of hypoxia signaling that regulates nonsmall-cell lung cancer development.

Expression of Aquaporin-1 and Aquaporin-5 in a Rat Model of High-Altitude Pulmonary Edema and the Effect of Hyperbaric Oxygen Exposure.

Objective:To investigate the therapeutic roles of hyperbaric oxygen exposure on high-altitude pulmonary edema and to determine whether aquaporin-1 and aquaporin-5 were involved in the pathogenesis of HAPE in rats.Methods:Rats were divided into 5 groups: The control group, the HAPE group (HAPE model), the HBO group (hyperbaric oxygen exposure), the NBO group (normobaric oxygen exposure), and the NA group (normal air exposure). Western blot and real-time PCR were used to analyze the pulmonary expressions of AQP1 and AQP5. The wet-to-dry (W/D) weight ratio and the morphology of the lung were also examined.Results:The lung W/D weight ratio in the HAPE group was increased compared with the control group. The injury score in the HBO group was noticeably lower than that in the control group. The mRNA and proteins expressions of AQP1 and AQP5 were significantly downregulated in the HAPE group.Conclusions:Oxygen exposure alleviated high-altitude hypobaric hypoxia-induced lung injury in rats. Additionally, HBO therapy had significant advantage on interstitial HAPE.

Toll-Like Receptor-4-Mediated Inflammation is Involved in Intermittent Hypoxia-Induced Lung Injury.

Intermittent hypoxia (IH) is a recognized risk factor for multiple organs damage, resulting in lung injury. Its pathophysiology is still poorly understood. Toll-like receptor 4 (TLR4) signaling plays a critical role in host immune response to invading pathogen and non-infectious tissue injury. The role of TLR4-mediated inflammation in IH-induced lung injury was investigated in this study. Lean adult male TLR4-deficient (TLR4-/-) mice and their controls (C57BL/6 mice) were exposed to either IH (FiO2 6-8% for 25s, 150s/cycle, 8h/day) or air (normoxic mice) for 6weeks. Animals were sacrificed after 6-week exposure, and the lung tissues were harvested for morphological and inflammatory analyses. The expression of TLR4 and nuclear factor kappa-B (NF-κB) P65 were examined by real-time quantitative polymerase chain reaction and immunohistochemical method. Serum cytokine levels of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were analyzed by enzyme-linked immunosorbent assay. IH induced morphological and inflammation changes in the lung. IH for 6weeks induced higher expression of TLR4 (C57BL/6-N vs C57BL/6-IH, P < 0.05) and resulted in higher release of TNF-α, IL-6 (P < 0.05), and NF-κB P65 (P < 0.05). These alterations were remitted by TLR4 deletion. TLR4-mediated inflammation plays an important role in the development of IH-induced lung injury in mice, possibly through mechanisms involving nuclear factor-κB. Targeting TLR4/NF-κB pathway could represent a further therapeutic option for sleep apnea patients.

Thyroid Function Modulates Lung Fluid and Alveolar Viscoelasticity in Mechanically Ventilated Rat

BackgroundMechanical ventilation (MV) is life saving; yet it may induce severe lung injury and lead to multisystem organ failure and death. Thyroid hormones (THs) promote alveolar fluid clearance and alleviates hypoxia-induced lung injury. Given that the mechanism involved in hypoxia-induced lung injury is different from that of ventilator-induced lung injury, we examined the effects of thyroid function on lung extravascular fluid (LF), aquaporin 5 (AQP 5) expression, and alveolar viscoelasticity (AVE) in mechanically ventilated rat. MethodsHypothyroid (hypo) and hyperthyroid (hyper) animals were generated by administration of metimazole and L-thyroxine, respectively. Lung injury was induced by high-tidal volume MV. The LF was estimated by lung wet weight-to-dry weight ratio assessment. Expression of AQP 5 was evaluated by western blotting and in situ immunohistochemistry. The AVE was judged by elastic lung pressure/volume curve recording. ResultsInjurious MV significantly reduced lung AQP 5 expression and altered LF and AVE in a thyroid function–dependent manner. Regardless of animals' ventilation mode, hyper state caused significant reductions in LF and lung AQP 5 protein. It also improved AVE irrespective of animals' ventilation mode. The effects of hypo condition on LF, AQP 5 expression, and AVE were in contrast to that of hyper state. ConclusionsThese data indicate that thyroid function has profound effects on LF, AQP 5, and AVE in mechanically ventilated lungs. Given that the effects of thyroidal status were as prominent as that of injurious MV, we suggest that thyroid function should be considered when patients are to be subjected to MV.

Heterozygous SOD2 deletion deteriorated chronic intermittent hypoxia-induced lung inflammation and vascular remodeling through mtROS-NLRP3 signaling pathway

Oxidative stress caused by chronic intermittent hypoxia (CIH) is the hallmark of obstructive sleep apnea (OSA). Among the first line of defense against oxidative stress is the dismutation of superoxide radicals, which in the mitochondria is carried out by manganese superoxide dismutase (SOD2). In this study, wild-type (WT) and SOD2-heterozygous knockout (SOD2+/−) mice were exposed to CIH or normoxic (Nor) conditions. After 4 weeks, pulmonary artery pressure was measured, and the mice were processed to harvest either serum for cytokine assays or lungs for flow cytometry and histopathological studies. Herein, we showed that heterozygous deletion of SOD2 markedly deteriorated pulmonary remodeling and increased the oxidative stress, especially promoted the infiltration of macrophages in the lungs of CIH mouse. Moreover, in the intermittent hypoxia (IH)-treated RAW264.7 cells, SOD2 knockdown increased the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation accompanied with the IL-1β elevation and caspase-1 activity. Additionally, mitochondrial ROS (mtROS) scavenger mito-TEMPO abolished NLRP3 inflammasome activation in IH-treated RAW264.7 cells. Collectively, our results supported that SOD2 contributed to the pathogenesis of CIH-induced lung remodeling. Meanwhile, SOD2 knockdown exacerbates oxidative damage through assembly and activation of NLRP3 inflammasome in macrophages. SOD2 may be a novel therapeutic target for CIH-induced pulmonary inflammation and arteriole remodeling.

Resveratrol Attenuates Intermittent Hypoxia-Induced Lung Injury by Activating the Nrf2/ARE Pathway.

Obstructive sleep apnea (OSA) is associated with lung injury. As a novel pathophysiological hallmark of OSA, chronic intermittent hypoxia (CIH) enhances apoptosis. The present study aims to evaluate the effect of resveratrol (Res) on CIH-induced lung apoptosis and inflammation in a rat model of CIH. Rats were randomly allocated to normoxia (control), CIH, and CIH + Res groups (n = 10 in each group). The CIH exposure duration was 12weeks. Rats in the CIH + Res group were additionally administered Res (50mgkg-1d-1). Inflammatory cytokine levels were detected by enzyme-linked immunosorbent assays (ELISAs). A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was conducted to evaluate the apoptosis rate. Bax, cleaved caspase-3, Nrf2 and HO-1 protein levels were detected by western blotting. The IL-6 and TNF-α levels in the serum and alveolar lavage fluid in the CIH group were markedly higher than those in the control group. The percentage of apoptotic cells in the CIH group was higher than that in the control group. Bax and cleaved caspase-3 protein levels were increased in the CIH group compared with those in the control group. Nrf2 and HO-1 protein levels were decreased in the CIH group compared with those in the control group (p < 0.05). Compared with the CIH group, rats in the CIH + Res group had lower percentages of apoptotic cells, lower IL-6, TNF-α, Bax and cleaved caspase-3 protein levels, and higher Nrf2 and HO-1 protein levels (p < 0.05). Res attenuates CIH-related inflammatory reactions and apoptosis in lung tissue by activating the Nrf2/ARE pathway.

Astragaloside IV ameliorates intermittent hypoxia-induced inflammatory dysfunction by suppressing MAPK/NF-κB signalling pathways in Beas-2B cells.

Intermittent hypoxia is a characteristic pathological change in obstructive sleep apnoea (OSA) that can initiate oxidative stress reaction and pro-inflammatory cytokine release. The purpose of this study was to assess the effect and protective mechanism of Astragaloside IV (AS-IV) in intermittent hypoxia-induced human lung epithelial Beas-2B cells. Human lung epithelial Beas-2B cells were exposed to intermittent hypoxia or normoxia in the absence or presence of AS-IV. MTT assay was performed to determine the cell viability. The levels of reactive oxygen species (ROS), lactate dehydrogenase (LDH), malonaldehyde (MDA), and superoxide dismutase (SOD) were measured to evaluate oxidative stress. The levels of cytokines interleukin (IL)-8, IL-1β, and IL-6 were evaluated by enzyme-linked immunosorbent assay and real-time PCR. The expression of Toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and nuclear transcription factor-kappa B (NF-κB) signalling pathways was analysed by western blot. The results showed that AS-IV significantly reduced the levels of ROS, LDH, MDA, IL-8, IL-1β, and IL-6, and increased the level of SOD in intermittent hypoxia-induced Beas-2B cells. It also suppressed the phosphorylation of MAPKs, including P38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, and inhibited the activation of the NF-κB signalling pathway by reducing the phosphorylation of IκBα and p65. AS-IV attenuates inflammation and oxidative stress by inhibiting TLR4-mediated MAPK/NF-κB signalling pathways in intermittent hypoxia-induced Beas-2B cells.

Netrin-1 Dampens Hypobaric Hypoxia-Induced Lung Injury in Mice.

Background: This study aimed to explore the effects of netrin-1 on hypobaric hypoxia-induced lung injury in mice. Methods: We exposed 6-8-week-old C57BL/6 mice to hypobaric stress at 340 mmHg for 30 minutes followed by 260 mmHg for different periods (6, 12, 18, and 24 hours) to observe the severity of lung injury (O2 concentration, 21%; 54.6 mmHg). The wet/dry weight ratio and protein leakage from the mouse lung were used to determine the suitable exposure time. Netrin-1 was injected into the tail vein of mice before 18-hour decompression. Inflammatory cytokines, lung injury scores, and activity of nuclear factor κB were evaluated. The expression of apoptosis-related proteins was also examined. Results: Protein concentration in the bronchoalveolar lavage fluid was significantly higher in the 18-hour group (p < 0.05). Pulmonary pathology revealed neutrophil infiltration, alveolar septum thickening, and tissue edema. Injury score and macrophage inflammatory protein 2 levels were also increased. Intrinsic apoptosis pathway was activated. Hypoxia decreased the expression of Bcl2 protein, the number of active caspase-3-stained cells, and UNC5HB receptors. Pretreatment with netrin-1 reduced protein leakage, inhibited neutrophil migration, lowered the injury score, attenuated apoptosis, and increased UNC5HB receptor expression. Conclusion: Netrin-1 dampens hypobaric hypoxia-induced lung injury by inhibiting neutrophil migration and attenuating apoptosis.

Uridine as a protector against hypoxia-induced lung injury

The effect of the activation of the mitochondrial ATP-dependent potassium channel (mitoKATP) on the ultrastructure of rat lung in acute hypoxic hypoxia (7% of oxygen in nitrogen, exposure 30 min) was studied. It was shown that uridine, a precursor of the mitoKATP activator UDP, exerted a protective effect against hypoxic damage to the lung. The administration of uridine to animals prior to hypoxia decreased the number of mitochondria with altered ultrastructure and prevented the hypoxia-induced mitochondrial swelling. Uridine also protected the epithelial, interstitial and endothelial layers of the air–blood barrier from the hypoxia-induced hyperhydration. The protective action of uridine against hypoxia-induced lung injury was eliminated by the selective blocker of mitoKATP 5-hydroxydecanoate. These data suggest that one of the mechanisms of the positive effect of uridine is related to the activation of the mitoKATP channel, which, according to the literature and our data, is involved in the protection of tissues from hypoxia and leads to adaptation to it. A possible role of uridine in the maintenance of the mitochondrial structure upon hypoxia-induced lung injury and the optimization of oxygen supply of the organism is discussed.

Pharmacological inhibition of β-catenin prevents EndMT in vitro and vascular remodeling in vivo resulting from endothelial Akt1 suppression

Endothelial to mesenchymal transition (EndMT), where endothelial cells acquire mesenchymal characteristics has been implicated in several cardiopulmonary, vascular and fibrotic diseases. The most commonly studied molecular mechanisms involved in EndMT include TGFβ, Notch, interleukin, and interferon-γ signaling. As of today, the contributions of Akt1, an important mediator of TGFβ signaling and a key regulator of endothelial barrier function to EndMT remains unclear. By using the ShRNA based gene silencing approach and endothelial-specific inducible Akt1 knockdown (ECKOAkt1) mice, we studied the role of Akt1 in EndMT in vitro and pathological vascular remodeling in vivo. Stable, Akt1 silenced (ShAkt1) human microvascular endothelial cells (HMECs) indicated increased expression of mesenchymal markers such as N-cadherin and α-SMA, phosphorylation of Smad2/3, cellular stress via activation of p38 MAP Kinase and the loss of endothelial nitric oxide synthase (eNOS) accompanied by a change in the morphology of HMECs in vitro and co-localization of endothelial and mesenchymal markers promoting EndMT in vivo. EndMT as a result of Akt1 loss was associated with increased expression of TGFβ2, a potent inducer of EndMT and mesenchymal transcription factors Snail1, and FoxC2. We observed that hypoxia-induced lung vascular remodeling is exacerbated in ECKOAkt1 mice, which was reversed by pharmacological inhibition of β-catenin. Thus, we provide novel insights into the role of Akt1-mediated β-catenin signaling in EndMT and pathological vascular remodeling, and present β-catenin as a potential target for therapy for various cardiopulmonary diseases involving vascular remodeling.

Hypoxia-induced Slug SUMOylation enhances lung cancer metastasis

BackgroundThe Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis.MethodsThe Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients.ResultsSlug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130–212 and 33–129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients.ConclusionsUbc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC.

Sinomenine Attenuates Chronic Intermittent Hypoxia-Induced Lung Injury by Inhibiting Inflammation and Oxidative Stress.

BackgroundIn the present study, we aimed to investigate the effects of sinomenine (SIN) on chronic intermittent hypoxia (CIH)- induced lung injury in rats, and to explore the underlying mechanisms.Material/MethodsTo perform the investigation, a CIH rat model was established. ELISA assay was applied to detect the level of inflammatory cytokines. Oxidative stress bio-markers (MDA, SOD, and CAT) were determined in lung tissues. In addition, the expression level of NADPH oxidase 2 (Nox2) was analyzed by Western blotting and qRT-PCR, respectively.ResultsThe results showed that compared with other groups, more obvious pulmonary pathological changes were observed in the CIH group. The level of inflammatory cytokines in the CIH group was markedly higher than that in the control and Con-S groups. Compared with the control and Con-S groups, oxidative stress was notably increased in the CIH group. Expression of Nox2 was also increased in the CIH group. The effects caused by CIH in rats were attenuated by SIN treatment.ConclusionsSIN can reverse chronic intermittent hypoxia-induced lung injury through inhibiting inflammation and oxidative stress.

Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3–6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

HIF and HOIL-1L–mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α1-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells (CreSPC/HOIL-1Lfl/fl ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α1-Na,K-ATPase construct bearing an S18A (α1-S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α1-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of CreSPC/HOIL-1Lfl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury.

Role of reactive oxygen species in hypoxia-induced non-small cell lung cancer migration

Objective: To explore reactive oxygen species (ROS) generation and its role on A549 cell migration under hypoxic condition. Methods: Human non-small cell lung cancer (NSCLC) cell line A549 was incubated in a hypoxic environment (1%O(2), hypoxia group) or in a normoxic environment (21%O(2), normoxia group). The generation of ROS was measured by flow cytometry. The cell motility of A549 cells was detected by Transwell assay. The protein levels of protein kinase B (AKT), p38 mitogen-activated protein kinase (p38) were determined by Western blot analysis. Results: After 16 h hypoxic treatment, the migration of A549 cells in hypoxia group was significantly more than that of normoxia group [(85±10) vs (56±7) per lower magnification, P<0.001]. Besides, the generation of ROS was in a time-depended manner in hypoxia group. The ROS level was increased with the prolonged hypoxia time. It was significantly higher at 24 h than that in normoxia group [(273±4)% vs (102±6)%, P<0.001]. The migrated cells in hypoxia group co-treated with 2 mmol/L NAC for 16 h were less than that with hypoxic treatment alone [(47±13) vs (105±14) per lower magnification, P=0.011]. Meanwhile, the phosphorylation of AKT and p38 increased after 12 h hypoxic treatment in hypoxia group, however, 2 mmol/L NAC co-treatment attenuated this effect. Furthermore, inhibition of phosphorylated AKT with 0.1 μmol/L allosteric AKT inhibitor (MK-2206) in hypoxia group for 16 h reversed the hypoxia-induced A549 cell migration. The migrated cells in hypoxia+ MK-2206 group were significantly less than that in hypoxia group [(155±21) vs (249±32) per lower magnification, P<0.001]. Conclusions: Hypoxia increases the generation of ROS in A549, resulting from oxidative stress under hypoxia. The increased ROS level promotes cell motility through the activation of AKT.

Role of Cyclooxygenase-2 on Intermittent Hypoxia-Induced Lung Tumor Malignancy in a Mouse Model of Sleep Apnea

An adverse role for obstructive sleep apnea (OSA) in cancer epidemiology and outcomes has recently emerged from clinical and animal studies. In animals, intermittent hypoxia (IH) mimicking OSA promotes tumor malignancy both directly and via host immune alterations. We hypothesized that IH could potentiate cancer aggressiveness through activation of the cyclooxygenase-2 (COX-2) pathway and the concomitant increases in prostaglandin E2 (PGE2). The contribution of the COX-2 in IH-induced enhanced tumor malignancy was assessed using celecoxib as a COX-2 specific inhibitor in a murine model of OSA bearing Lewis lung carcinoma (LLC1) tumors. Exposures to IH accelerated tumor progression with a tumor associated macrophages (TAMs) shift towards a pro-tumoral M2 phenotype. Treatment with celecoxib prevented IH-induced adverse tumor outcomes by inhibiting IH-induced M2 polarization of TAMs. Furthermore, TAMs isolated from IH-exposed mice treated with celecoxib reduced the proliferation of LLC1 naïve cells, while the opposite occurred with placebo-treated IH-exposed mice. Finally, in vitro IH exposures of murine macrophages and LLC1 cells showed that both cell types increased PGE2 release in response to IH. These results suggest a crucial role for the COX-2 signaling pathway in the IH-exacerbated malignant processes, and designate macrophages and lung adenocarcinoma cells, as potential sources of PGE2.

Daxx inhibits hypoxia-induced lung cancer cell metastasis by suppressing the HIF-1α/HDAC1/Slug axis.

Hypoxia is a major driving force of cancer invasion and metastasis. Here we show that death domain-associated protein (Daxx) acts to negatively regulate hypoxia-induced cell dissemination and invasion by inhibiting the HIF-1α/HDAC1/Slug pathway. Daxx directly binds to the DNA-binding domain of Slug, impeding histone deacetylase 1 (HDAC1) recruitment and antagonizing Slug E-box binding. This, in turn, stimulates E-cadherin and occludin expression and suppresses Slug-mediated epithelial–mesenchymal transition (EMT) and cell invasiveness. Under hypoxic conditions, stabilized hypoxia-inducible factor (HIF)-1α downregulates Daxx expression and promotes cancer invasion, whereas re-expression of Daxx represses hypoxia-induced cancer invasion. Daxx also suppresses Slug-mediated lung cancer metastasis in an orthotopic lung metastasis mouse model. Using clinical tumour samples, we confirmed that the HIF-1α/Daxx/Slug pathway is an outcome predictor. Our results support that Daxx can act as a repressor in controlling HIF-1α/HDAC1/Slug-mediated cancer cell invasion and is a potential therapeutic target for inhibition of cancer metastasis.

Intermittent Hypoxia Contributes to the Lung Damage by Increased Oxidative Stress, Inflammation, and Disbalance in Protease/Antiprotease System.

Intermittent hypoxia as a surrogate of obstructive sleep apnea is associated with different cardiovascular complications. However, the effects of intermittent hypoxia on the lung tissue are less known. Therefore, the aim of our present study was to investigate if intermittent hypoxia may influence oxidative stress, inflammation, and protease/antiprotease system in the lung. Additionally, potential protective properties of anti-inflammatory and anti-oxidative drugs have been evaluated. 32 mice were divided into four groups: (1) intermittent hypoxia, (2) intermittent hypoxia with infliximab, (3) intermittent hypoxia with L-glutathione, and (4) normoxia. After 4weeks, lungs and blood were collected. Levels of reactive oxygen species in the lung were calculated by L-O12-enhanced chemiluminescence. CD68-positive lung macrophages were detected by immunofluorescence. Concentrations of elastase and desmosine in lung and of alpha-1-antitrypsin in blood were calculated by means of enzyme-linked immunosorbent assay. Compared to a control, intermittent hypoxia augmented the release of free oxygen radicals, expression of CD68+ macrophages, and concentration of elastase in the lung tissue. Despite increased blood levels of protective alpha-1-antitrypsin, concentrations of desmosine-degradation product of elastin were higher versus control. The application of anti-inflammatory infliximab und anti-oxidative L-glutathione prevented at least partly the above-observed hypoxia-associated changes. Intermittent hypoxia contributes to the lung damage by increased oxidative stress, inflammation, and disbalance in protease/antiprotease system. Infliximab and L-glutathione may prevent adverse hypoxia-induced lung alternations.