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.     

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.     

Nephropreventing effect of hypoxia‐inducible factor 1α in a rat model of ischaemic/reperfusion acute kidney injury

Acute kidney injury (AKI) occurs in 5% of hospitalized patients and in 50% of sepsis patients with acute renal dysfunction. However, there have been no safe and effective therapeutic strategies. The hypoxia condition is closely related to renal injury and function under AKI. As hypoxia‐inducible factor 1α (HIF‐1α) is critical for the cellular response to hypoxia, we investigated the protective effect of HIF‐1α in a rat AKI model. We found that HIF‐1α injection improved the survival of rat with AKI, and the level of creatinine and blood urea nitrogen (BUN) was also increased. Our data showed that HIF‐1α treatment significantly alleviated ischaemic/reperfusion injury to kidney tubules and nephrocytes. We also found the downstream factors, such as EPOR, VEGF, and PHD3, were also upregulated by HIF‐1α. Finally, it was observed that HIF‐1α treatment also increased the percentage of adult resident progenitor cells (ARPC) in vitro and in vivo. In conclusion, HIF‐1α plays a protective role in the ischaemic AKI model through stimulating the proliferation of ARPC, and our study provided a potential therapeutic strategy for AKI.     

Icaritin inhibits the invasion and epithelial‐to‐mesenchymal transition of glioblastoma cells by targeting EMMPRIN via PTEN/AKt/HIF‐1α signalling

Icaritin, a hydrolytic product of icariin from the Epimedium genus, exerts anti‐tumour effects on a variety of tumour cell types, mainly by inhibiting cell proliferation and inducing apoptosis. However, little is known about the role of icaritin in cancer invasion and epithelial‐to‐mesenchymal transition (EMT). In the present study, the glioblastoma (GBM) cell line U87MG was used as a model to investigate the effects of icaritin on the invasion and EMT of cancer cells. The results showed that icaritin significantly inhibited the invasion and EMT of GBM cells by targeting extracellular matrix metalloproteinase (EMMPRIN). Furthermore, the findings strongly indicate that the modulatory effect of icaritin on EMMPRIN is mediated via the PTEN/Akt/HIF‐1α signalling pathway. The data provide the first experimental evidence of the inhibitory effect of icaritin on cancer cell invasion and EMT, thus highlighting the potential of icaritin to be employed as a promising anti‐cancer agent in the treatment of GBM.     

Comparative assessment of HIF‐1α and Akt responses in human lung and skin cells exposed to benzo[α]pyrene: Effect of conditioned medium from pre‐exposed primary fibroblasts

Exposure to atmospheric pollutants has been accused for many adverse health effects. Benzo[α]pyrene (Β[α]Ρ) in particular, the most extensively studied member of pollutants, is implicated in both cancer initiation and promotion. In the present study, we compared the effects of noncytotoxic doses of Β[α]Ρ, between human skin and lung epithelial cells A431 and A549, respectively, focusing on Akt kinase and HIF‐1α, as it is well known that these proteins are upregulated in various human cancers promoting survival, angiogenesis and metastasis of tumor cells. Also, taking into consideration that fibroblasts are involved in cancer progression, we tested the possible modulation of epithelial cell response by paracrine factors secreted by Β[α]Ρ‐treated fibroblasts. Low doses of Β[α]Ρ were found to enhance epithelial cell proliferation and upregulate both Akt kinase and HIF‐1α, with A549 cells exhibiting a more sustained profile of upregulation. It is to notice that, the response of HIF‐1α was remarkably early, acting as a sensitive marker in response to airborne pollutants. Also, HIF‐1α was induced by Β[α]Ρ in both lung and skin fibroblasts indicating that this effect may be conserved throughout different cell types and tissues. Interestingly however, the response of both proteins was differentially modified upon treatment with conditioned medium from Β[α]Ρ‐exposed fibroblasts. This is particularly evident in A459 cells and confirms the critical role of intercellular and paracrine factors in the modulation of the final response to an extracellular signal. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1103–1112, 2016.     

Endotoxin‐induced acute lung injury in mice is protected by 5,7‐dihydroxy‐8‐methoxyflavone via inhibition of oxidative stress and HIF‐1α

Up to date, the morbidity and mortality rates of acute lung injury (ALI) still rank high among clinical illnesses. Endotoxin, also called lipopolysaccharide (LPS), induced sepsis is the major cause for ALI. Beneficial biological effects, such as antioxidation, anti‐inflammation, and neuroprotection was found to express by 5,7‐dihydroxy‐8‐methoxyflavone (DHMF). The purpose of present study was to investigate the potential protective effects of DHMF and the possibile mechanisms involved in LPS‐induced ALI. In our experimental model, ALI was induced in mice by intratracheal injection of LPS, and DHMF at various concentrations was injected intraperitoneally for 30 min prior to LPS administration. Pretreatment with DHMF inhibited not only the histolopatholgical changes occurred in lungs but also leukocytes infiltration in LPS‐induced ALI. Decreased activity of antioxidative enzymes (AOE) such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) caused by LPS was reversed by DHMF. LPS‐induced lipid peroxidation HIF‐1α accumulation, NF‐κB phosphorylation, and IκBα degradation were all inhibited by DHMF. In addition, LPS‐induced expression of proinflammatory mediators such as TNF‐α and IL‐1β were also inhibited by 5,7‐dihydroxy‐8‐methoxyflavone. These results suggested that the protective mechanisms of DHMF on endotoxin‐induced ALI might be via up‐regulation of antioxidative enzymes, inhibition of NFκB phosphorylation, and HIF‐1α accumulation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1700–1709, 2016.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Identification of small molecule estrogen‐related receptor α–specific antagonists and homology modeling to predict the molecular determinants as the basis for selectivity over ERRβ and ERRγ

The estrogen‐related receptor α (ERRα) was one of the first orphan receptors identified through a search for genes encoding proteins related to the steroid nuclear receptor, Estrogen Receptor α (ERα). The physiological role of ERRα has not yet been established nor has a natural ligand been elucidated. Importantly, research indicates that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders. A homogeneous time‐resolved fluorescence (HTRF) assay has been developed to screen for ERRα‐specific antagonists. This assay uses the ERR ligand binding domain and the coactivator interaction domain of Proliferator‐activated Receptor γ Coactivator‐1α (PGC‐1α) to examine the ability of compounds to antagonize the constitutive interaction between ERRα and the coactivator. A dissociation‐enhanced lanthanide fluorescence immunoassay (DELFIA) was also created to counter screen compounds identified in the HTRF screen. Here we report the discovery of high‐affinity ERRα subtype selective antagonists. Additionally, a homology model of ERRα in an antagonist conformation has been developed and after subsequent docking studies, we offer a model showing the molecular determinants that suggest why our novel tri‐cyclic antagonist, N‐[(2Z)‐3‐(4,5‐dihydro‐1,3‐thiazol‐2‐yl)‐1,3‐thiazolidin‐2‐yl idene]‐5 H dibenzo[a,d][7]annulen‐5‐amine, binds to ERRα with high affinity but does not bind to either ERRβ or ERRγ. Drug Dev Res 69:203–218, 2008. © 2008 Wiley‐Liss, Inc.     

Peroxisome proliferator‐activated receptor‐γ coactivator‐1α in muscle links metabolism to inflammation

1. In higher eukaryotes, metabolism and immunity are tightly coupled. However, whereas in evolutionary terms a compromised immune response due to undernourishment has been the predominant problem, the inflammatory response to obesity and other lifestyle‐associated diseases has increased in relevance in Western societies in the past 100 years. 2. Traditionally, fat tissue has been considered as the major source of pro‐inflammatory secreted factors in these pathologies. However, in recent years the contribution of other tissues to disease‐causing chronic inflammation has been increasingly appreciated. 3. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) is one of the key regulatory factors in active skeletal muscle. Aberrant expression of PGC‐1α in inactive muscle fibres could be linked to a sedentary lifestyle, persistent systemic inflammation and a higher risk for many chronic diseases. Accordingly, modulation of PGC‐1α activity in skeletal muscle may have a broad range of therapeutic effects. Here, recent advances in the understanding of the role of muscle PGC‐1α in health and disease are reviewed.     

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.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

β‐LAPachone ameliorates doxorubicin‐induced cardiotoxicity via regulating autophagy and Nrf2 signalling pathways in mice

β‐LAPachone (B‐LAP) is a naphthoquinone that possesses antioxidant properties. In the present investigation, the protective effect of B‐LAP against doxorubicin (DOX)‐induced cardiotoxicity was examined in mice. Thirty‐five mice were divided into 5 groups: control group, B‐LAP (5 mg/kg) group, DOX (15 mg/kg) group, DOX+B‐LAP (2.5 mg/kg) group and DOX+B‐LAP (5 mg/kg) group. B‐LAP was administered orally for 14 days of experimental period. A single dose of DOX (15 mg/kg) was injected intraperitoneally on day 3. Cardiac function, histoarchitecture, indices of oxidative stress and circulating markers of cardiac injury were examined. B‐LAP (5 mg/kg) decreased serum levels of lactate dehydrogenase (LDH), creatine kinase MB (CK‐MB) and cardiac troponin I (cTnI), and ameliorated cardiac histopathological alterations. In addition to increasing cellular NAD⁺/NADH ratio, B‐LAP up‐regulated the cardiac levels of SIRT1, beclin‐1, p‐LKB1 and p‐AMPK, and reduced the cardiac levels of p‐mTOR, interleukin (IL)‐1β, TNF (tumour necrosis factor)‐α and caspase‐3. B‐LAP also elevated the nuclear accumulation of Nrf2 and simultaneously up‐regulated the protein levels of haem oxygenase (HO‐1) and glutathione S‐transferase (GST) in the hearts of DOX mice. While B‐LAP reduced malondialdehyde concentrations in heart of DOX‐treated mice, it further promoted the activities of cardiac superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT).In accordance with increased cell survival, B‐LAP significantly improved the cardiac function of DOX mice. Collectively, these findings underline the protective potential of B‐LAP against DOX‐induced cardiotoxicity by regulating autophagy and AMPK/Nrf2 signalling pathway in mice.     

Growth inhibitory effect of β‐eudesmol on cholangiocarcinoma cells and its potential suppressive effect on heme oxygenase‐1 production,STAT1/3 activation,and NF‐κB downregulation

Cholangiocarcinoma (CCA) is a progressively fatal form of cancer originating from the malignant transformation of hepatic biliary cholangiocytes. The present study reports for the first time in vitro growth inhibitory activities of β‐eudesmol, the bioactive sesquiterpenoid present in the rhizome of Atractylodes lancea (Thunb) DC., with respect to its underlying potential effects on heme oxygenase‐1 (HO‐1) production, STAT1/3 phosphorylation, and NF‐κB protein expression in human CCA cell line CL‐6. The cytotoxic effect of β‐eudesmol on CL‐6 cells was evaluated by MTT assay using normal human embryonic fibroblast (OUMS) as a control cell line. Results indicated that β‐eudesmol exhibited selective cytotoxicity towards CL‐6 compared to OUMS with mean (±SD) IC₅₀ (concentration that inhibits cell growth by 50%) values of 166.75 ± 3.69 and 240.01 ± 16.54 μmol/L, respectively. In addition, it also significantly suppressed colony forming and wound healing ability of CL‐6 cells in a concentration‐dependent manner. Western blot analysis indicated that β‐eudesmol treatment resulted in significant suppression of HO‐1 production in CL‐6 cells. Its inhibitory effects on the phosphorylation of STAT1/3 proteins and expression of NF‐κB (p65 and p50) proteins were concentration‐dependent. Taken together, these results suggest that β‐eudesmol exerts significant growth inhibitory activity on CL‐6 cells that may be linked to its inhibitory effect on the production of HO‐1, phosphorylation of STAT1/3, and expression of major NF‐κB proteins.