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

Andrographolide is a potent anti‐inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET‐1) is an endothelium‐derived vasoconstrictor with pro‐inflammatory properties secreted in response to hypoxia. Mitogen‐activated protein kinase phosphatase 5 (MKP‐5) is a dual‐specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia‐induced HIF‐1α expression and ET‐1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP‐5 plays in andrographolide's inhibition of hypoxia‐induced expression of HIF‐1α and ET‐1. Hypoxic conditions were created using the hypoxia‐mimetic agent CoCl₂. Andrographolide enhanced HO‐1 and MKP‐5 expression and cellular cGMP content in addition to inhibiting hypoxia‐induced ROS generation. Concomitantly, the HO‐1 byproduct CO and the cGMP analogue 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) increased MKP‐5 expression, and pretreatment with CO and 8‐Br‐cGMP inhibited hypoxia‐induced HIF‐1α and ET‐1 expression. Transfection of HO‐1 siRNA or pretreatment with the HO‐1 inhibitor ZnPP‐9 or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide‐induced MKP‐5 expression. Moreover, silencing MKP‐5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia‐induced p38 MAPK activation and HIF‐1α expression. The inhibition of hypoxia‐induced HIF‐1α and ET‐1 expression by andrographolide is likely associated with HO‐1/CO/cGMP/MKP‐5 pathways, which is involved in inhibiting hypoxia‐induced p38 MAPK activation.     

Chronic treatment with tempol does not significantly ameliorate renal tissue hypoxia or disease progression in a rodent model of polycystic kidney disease

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Silencing of hypoxia inducible factor‐1α by RNA interference inhibits growth of SK‐NEP‐1 Wilms tumour cells in vitro,and suppresses tumourigenesis and angiogenesis in vivo

Wilms tumour is the most common tumour of the pediatric kidney. Elevation of hypoxia‐inducible factor 1α (HIF‐1α) has been detected in 93% to 100% of human Wilms tumour specimens, suggesting a potential value of HIF‐1α as a therapeutic target for Wilms tumour. In the present study, a stable HIF‐1α‐silenced Wilms tumour cell strain was established by introducing HIF‐1α short‐hairpin RNA (shRNA) into SK‐NEP‐1 cells. Silencing of HIF‐1α significantly reduced single‐cell growth capacity, suppressed proliferation and arrested cell cycle of SK‐NEP‐1 cells. In addition, reduction of HIF‐1α expression induced apoptosis in SK‐NEP‐1 cells, which was accompanied by increased levels of cleaved caspase‐3, cleaved poly (ADP‐ribose) polymerase (PARP) and Bax as well as downregulation of Bcl‐2 in the cells. Furthermore, when inoculated subcutaneously in nude mice, HIF‐1α‐silenced SK‐NEP‐1 cells displayed retarded tumour growth and impaired tumour angiogenesis. In summary, the findings of this study suggest that HIF‐1α plays a critical role in the development of Wilms tumour, and it may serve as a candidate target of gene therapy for Wilms tumour.     

Hypoxic preconditioning facilitates acclimatization to hypobaric hypoxia in rat heart

Objectives Acute systemic hypoxia induces delayed cardioprotection against ischaemia‐reperfusion injury in the heart. As cobalt chloride (CoCl₂) is known to elicit hypoxia‐like –responses, it was hypothesized that this chemical would mimic the preconditioning effect and facilitate acclimatization to hypobaric hypoxia in rat heart. Methods Male Sprague‐Dawley rats treated with distilled water or cobalt chloride (12.5 mg Co/kg for 7 days) were exposed to simulated altitude at 7622 m for different time periods (1, 2, 3 and 5 days). Key findings Hypoxic preconditioning with cobalt appreciably attenuated hypobaric hypoxia‐induced oxidative damage as observed by a decrease in free radical (reactive oxygen species) generation, oxidation of lipids and proteins. Interestingly, the observed effect was due to increased expression of the antioxidant proteins hemeoxygenase and metallothionein, as no significant change was observed in antioxidant enzyme activity. Hypoxic preconditioning with cobalt increased hypoxia‐inducible factor 1α (HIF‐1α) expression as well as HIF‐1 DNA binding activity, which further resulted in increased expression of HIF‐1 regulated genes such as erythropoietin, vascular endothelial growth factor and glucose transporter. A significant decrease was observed in lactate dehydrogenase activity and lactate levels in the heart of preconditioned animals compared with non‐preconditioned animals exposed to hypoxia. Conclusions The results showed that hypoxic preconditioning with cobalt induces acclimatization by up‐regulation of hemeoxygenase 1 and metallothionein 1 via HIF‐1 stabilization.     

67‐kDa Laminin receptor contributes to hypoxia‐induced migration and invasion of trophoblast‐like cells by mediating matrix metalloproteinase‐9

Insufficient trophoblast invasion often occurs in patients experiencing preeclampsia. The 67‐kDa laminin receptor (LR1) is a multifunctional protein that binds to laminin and interacts with the extracellular matrix. We recently demonstrated that LR1 is implicated in trophoblast migration and invasion. However, whether LR1 is involved in hypoxia‐mediated trophoblastic invasion remains unclear and requires further investigation. This study demonstrates that two trophoblast‐like cell lines (JEG3 and BeWo cells) cultured at 3% oxygen exerted enhanced migratory and invasive capabilities as compared with their counterparts exposed to 20% oxygen. LR1 expression was increased in hypoxic JEG3 cells but decreased after transfection with hypoxia‐inducible factor 1 alpha (HIF‐1α) specific siRNA. Moreover, shRNA targeting LR1 mRNA significantly inhibited hypoxia‐induced increase in matrix metalloproteinase (MMP)‐9 activity in JEG3 cells. Forced overexpression of LR1 augmented JEG3 cell migration and invasion, and enhanced MMP‐9 expression and activity. Additionally, the blockade of the MMP‐9 effect with its neutralizing antibody reduced LR1 elevation‐promoted trophoblastic invasion. In summary, this study demonstrates that LR1 contributes to hypoxia‐induced migration and invasion of trophoblast cells at least partly by mediating MMP‐9 in vitro.     

Synthesis and evaluation of 2‐, 4‐, 5‐substituted nitroimidazole‐iminodiacetic acid‐99mTc(CO)3 complexes to target hypoxic tumors

Determination of hypoxia in tumor is an important problem in the clinical management of cancer. Towards this, a series of differently substituted nitroimidazoles, viz. 2‐nitro, 4‐nitro and 5‐nitroimidazole iminodiacetic acid (IDA) derivatives were synthesized and radio‐labeled with a [^99mTc(CO)₃(H₂O)₃]⁺ core. The corresponding ^185/187Re(CO)₃ analogue of 2‐nitroimidazole‐IDA‐^99mTc(CO)₃ complex was also prepared and characterized to elucidate the mode of bonding between the ligand and the M(CO)₃ core (M=^99mTc, ^185/187Re). All the three nitroimidazole‐IDA‐^99mTc(CO)₃ complexes could be prepared in over 95% yield determined by HPLC. The three complexes were then evaluated in a suitable animal model bearing tumor. Though the in vivo accumulation of complexes in hypoxic tissue is governed by factors such as lipophilicity, charge, etc., the variation in accumulation in hypoxic tissue, in the present case, could be explained by considering the reported values of single electron reduction potential of the respective nitroimidazoles. Among the three derivatives studied, the 2‐nitroimidazole‐IDA‐^99mTc(CO)₃ complex produced the best result followed by the 5‐nitroimidazole complex. Copyright © 2010 John Wiley & Sons, Ltd.     

Hypoxia inducible factor‐1α inhibition produced anti‐allodynia effect and suppressed inflammatory cytokine production in early stage of mouse complex regional pain syndrome model

Complex regional pain syndrome (CRPS) is related to microcirculation impairment associated with tissue hypoxia and peripheral cytokine overproduction in the affected limb. Previous studies suggest that the pathogenesis involves hypoxia inducible factor‐1α (HIF‐1α) and exaggerated regional inflammatory response. 1‐methylpropyl 2‐imidazolyl disulfide (PX‐12) acts as the thioredoxin‐1 (Trx‐1) inhibitor and decreases the level of HIF‐1α, and can rapidly be metabolized for Trx‐1 redox inactivation. This study hypothesized that PX‐12 can decrease the cytokine production for nociceptive sensitization in the hypoxia‐induced pain model. CD1 mice weighing around 30 g were used. The animal CRPS model was developed via the chronic post‐ischaemic pain (CPIP) model. The model was induced by using O‐rings on the ankles of the mice hind limbs to produce 3‐h ischaemia–reperfusion injury on the paw. PX‐12 (25 mg/kg, 5 mg/kg) was given through tail vein injection immediately after ischaemia. Animal behaviour was tested using the von Frey method for 7 days. Local paw skin tissue was harvest from three groups (control, 5 mg/kg, 25 mg/kg) 2 h after injection of PX‐12. The protein expression of interleukin‐1β (IL‐1β) and HIF‐1α was analysed with the Western blotting method. Mice significantly present an anti‐allodynia effect in a dose‐related manner after the PX‐12 administration. Furthermore, PX‐12 not only decreased the expression of HIF‐1α but also decreased the expression of IL‐1β over the injured palm. This study, therefore, shows the first evidence of the anti‐allodynia effect of PX‐12 in a CPIP animal model for pain behaviour. The study concluded that inhibition of HIF‐1α may produce an analgesic effect and the associated suppression of inflammatory cytokine IL‐1β in a CPIP model.     

Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia‐inducible factors HIF1α and HIF2α

Chemotherapy resistance is a major contributor to poor treatment responses and tumour relapse, the development of which has been strongly linked to the action of cancer stem cells (CSCs). Mounting evidence suggests that CSCs are reliant on low oxygen conditions and hypoxia‐inducible factors 1α and 2α (HIF1α and HIF2α) to maintain their stem cell features. Research in the last decade has begun to clarify the functional differences between the two HIFα subtypes (HIFαs). Here, we review and discuss these differences in relation to CSC‐associated drug resistance. Both HIFαs contribute to CSC survival but play different roles –HIF1α being more responsible for survival functions and HIF2α for stemness traits such as self‐renewal – and are sensitive to different degrees of hypoxia. Failure to account for physiologically relevant oxygen concentrations in many studies may influence the current understanding of the roles of HIFαs. We also discuss how hypoxia and HIFαs contribute to CSC drug resistance via promotion of ABC drug transporters Breast cancer resistance protein (BCRP), MDR1, and MRP1 and through maintenance of quiescence. Additionally, we explore the PI3K/AKT cell survival pathway that may support refractory cancer by promoting CSCs and activating both HIF1α and HIF2α. Accordingly, HIF1α and HIF2α inhibition, potentially via PI3K/AKT inhibitors, could reduce chemotherapy resistance and prevent cancer relapse.     

Fluorine‐18 radiolabeling of a nitrophenyl sulfoxide and its evaluation in an SK‐RC‐52 model of tumor hypoxia

The significance of imaging hypoxia with the positron emission tomography ligand [¹⁸F]FMISO has been demonstrated in a variety of cancers. However, the slow kinetics of [¹⁸F]FMISO require a 2‐h delay between tracer administration and patient scanning. Labeled chloroethyl sulfoxides have shown faster kinetics and higher contrast than [¹⁸F]FMISO in a rat model of ischemic stroke. However, these nitrogen mustard analogues are unsuitable for routine production and use in humans. Here, we report on the synthesis and in vitro and in vivo evaluation of a novel sulfoxide, which contains an ester moiety for hydrolysis and subsequent trapping in hypoxic cells. Non‐decay corrected yields of radioactivity were 1.18 ± 0.24% (n = 27, 2.5 ± 0.5% decay corrected radiochemical yield) based on K[¹⁸F]F. The radiotracer did not show any defluorination and did not undergo metabolism in an in vitro assay using S9 liver fractions. Imaging studies using an SK‐RC‐52 tumor model in BALB/c nude mice have revealed that [¹⁸F]1 is retained in hypoxic tumors and has similar hypoxia selectivity to [¹⁸F]FMISO. Because of a three times faster clearance rate than [¹⁸F]FMISO from normoxic tissue, [¹⁸F]1 has emerged as a promising new radiotracer for hypoxia imaging.     

Toxic effects of microcystin‐LR on the HepG2 cell line under hypoxic and normoxic conditions

Microcystins (MCs) are highly liver‐specific and evidenced as a liver tumor promoter. Oxidative stress is one of the most important toxicity mechanisms of MCs, which is tightly related to oxygen concentration. The effects of MCs on animals and cell lines in normoxia and the mechanisms have been well studied, but such effects in different oxygen conditions were still unclear. The aim of the present study was to explore the cellular response of the human hepatocellular carcinoma cell line (HepG2) to MC‐LR exposure under hypoxic (1% O₂) and normoxic (21% O₂) conditions. We examined cell viability, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity and gene expression posttoxin exposure. Cell viability was increased by MC‐LR in normoxia although decreased in hypoxia. MC‐LR markedly induced MMP loss under hypoxic condition but only slightly MMP loss under normoxic condition. SOD activity was significantly induced by MC‐LR in hypoxia, indicating prolonged oxidative stress. Inhibitory apoptosis protein (c‐IAP2) was significantly up‐regulated by MC‐LR under normoxic condition, suggesting that c‐IAP2 played an important role in the promotion of cell proliferation by MC‐LR. These results indicate that MC‐LR promotes cell proliferation under normoxic condition whereas induces cell apoptosis under hypoxic condition. Copyright © 2012 John Wiley & Sons, Ltd.