Hypoxia-inducible Factor-1α Levels Research Articles

Cyclophilin a represses reactive oxygen species generation and death of hypoxic non-small-cell lung cancer cells by degrading thioredoxin-interacting protein

ABSTRACT Cyclophilin A (cypA) is overexpressed in many types of carcinomas, including non-small-cell lung cancer (NSCLC). However, the effect of anoxia, a critical feature of the carcinoma cell microenvironment, on cypA expression in NSCLC is unknown. Here, formaldehyde-fixed and paraffin-embedded samples were collected from 60 subjects with NSCLC. The protein expression levels of cypA and hypoxia-inducible factor-1α (HIF-1α) were evaluated using immunohistochemistry. Kaplan–Meier analysis showed that subjects with high cypA expression had remarkably shorter progression-free survival than those with low cypA expression. Furthermore, cypA expression levels were significantly related to HIF-1α expression levels (Spearman’s correlation = 0.34, P < 0.0001). To further assess the effect of cypA, an anoxic carcinoma cell model was established. CypA expression was remarkably upregulated in H1299 and A549 cell lines under hypoxic conditions. Overexpression of cypA restored hypoxia-impaired cell growth and prevented reactive oxygen species (ROS) production and cell death in hypoxic A549 and H1299 cells. However, these phenotypes were not altered by the inactive R55A mutant of cypA. Mechanistic studies demonstrated that cypA can bind to and degrade the tumor suppressor protein TXNIP in H1299 and A549 cells. Restored TXNIP expression in cypA-overexpressed and hypoxic NSCLC cells led to increased ROS levels and apoptotic cell numbers and decreased cell growth compared with cypA-overexpressed and hypoxic NSCLC cells. These findings indicate that anoxia results in an increase in cypA expression in NSCLC. Additionally, cypA served as an oncogene during hypoxia by interacting with TXNIP.

Roxadustat, a Hypoxia-Inducible Factor 1α Activator, Attenuates Both Long- and Short-Term Alcohol-Induced Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a worldwide healthcare problem featured by inflammation, reactive oxygen species (ROS), and lipid dysregulation. Roxadustat is used for chronic kidney disease anemia treatment. As a specific inhibitor of prolyl hydroxylase, it can maintain high levels of hypoxia-inducible factor 1α (HIF-1α), through which it can further influence many important pathways, including the three featured in ALD. However, its effects on ALD remain to be elucidated. In this study, we used chronic and acute ALD mouse models to investigate the protective effects of roxadustat in vivo. Our results showed that long- and short-term alcohol exposure caused rising activities of serum transaminases, liver lipid accumulation, and morphology changes, which were reversed by roxadustat. Roxadustat-reduced fatty liver was mainly contributed by the reducing sterol-responsive element-binding protein 1c (SREBP1c) pathway, and enhancing β-oxidation through inducing peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) expression. Long-term alcohol treatment induced the infiltration of monocytes/macrophages to hepatocytes, as well as inflammatory cytokine expression, which were also blocked by roxadustat. Moreover, roxadustat attenuated alcohol caused ROS generation in the liver of those two mouse models mainly by reducing cytochrome P450 2E1 (CYP2E1) and enhancing superoxidase dismutase 1 (SOD1) expression. In vitro, we found roxadustat reduced inflammation and lipid accumulation mainly via HIF-1α regulation. Taken together, our study demonstrates that activation of HIF-1α can ameliorate ALD, which is contributed by reduced hepatic lipid synthesis, inflammation, and oxidative stress. This study suggested that roxadustat could be a potential drug for ALD treatment.

Early biomarkers of nephrotoxicity associated with the use of anti-VEGF drugs.

Anti-angiogenic anticancer drugs that block vascular endothelial growth factor (VEGF) can cause kidney damage. An early assessment of the risk of nephrotoxicity would allow the development of optimal treatment approaches and allow for relatively safer therapeutic regimens. The aim of this study was to assess the utility of neutrophilic gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), hypoxia inducible factor-1α (HIF-1α) and nephrin levels in urine as early biomarkers for the nephrotoxicity of anti-VEGF drugs. The study included 50 patients who received anti-VEGF drugs (aflibercept, bevacizumab or ramucirumab) for 8 weeks. The levels of KIM-1, NGAL, HIF-1α and nephrin in urine samples were determined by ELISA before treatment and after 1, 2, 4 and 8 weeks of treatment. To assess risk factors for nephrotoxicity, a logistic regression analysis was performed with the inclusion of the primary clinical and laboratory parameters. The primary outcome measure was a decrease in glomerular filtration rate (GFR) to <60 ml/min/1.73 m2 at 8 weeks, and nephrotoxicity resulting in discontinuation within 9 months. The primary outcome goal was achieved in 21 (42%) patients treated with anti-VEGF drugs. Increased NGAL, KIM-1, HIF-1α and nephrin levels in urine at 1 week of treatment predicted the development of nephrotoxicity. High sensitivity and specificity of these urinary biomarkers were established by ROC analysis: KIM-1 [area under the curve (AUC) 0.69], NGAL (AUC 0.7), HIF-1α (AUC 0.7) and nephrin (AUC 0.7). The unfavorable predictors of nephrotoxicity were an initial decrease in estimated GFR, a history of arterial hypertension, and an increase in urinary concentration KIM-1 OR of 1.1 [CI 95% 1.02-1.183], and HIF-1α OR of 5.6 [CI 95% 3.601-8.949] (P<0,05) at 1 and 2 weeks of treatment. Urinary NGAL, KIM-1, HIF-1α and nephrin are early biomarkers of nephrotoxicity following treatment with anti-VEGF anticancer drugs. The independent risk factors for nephrotoxicity are the initial decrease in GFR, arterial hypertension, and an increase in the concentration of KIM-1 and HIF-1α in the urine in the early stages of therapy.

Hypoxia‑induced mitophagy regulates proliferation, migration and odontoblastic differentiation of humandental pulp cells through FUN14 domain‑containing 1.

FUN14 domain-containing 1 (FUNDC1) is a receptor that has been previously reported to activate hypoxia-induced mitophagy. However, the potential role of FUNDC1 in the pathophysiology of dental pulp diseases remains unknown. Therefore, present study first collected tissue specimens from patients with pulpitis and from healthy individuals. The results of reverse transcription-quantitative PCR and immunohistochemical staining revealed markedly increased FUNDC1 and hypoxia-inducible factor-1α expression in pulpitis tissue specimens compared with those from healthy individuals. To provide a theoretical basis for the study of the occurrence, development and reparative mechanisms in the dental pulp after tissue injury, the present study then investigated the role of hypoxia-induced mitophagy in the regulation of proliferation, migration and odontoblastic differentiation in human dental pulp cells (HDPCs), in addition, to the possible involvement of FUNDC1. The surface markers and multipotent differentiation capabilities of HDPCs were performed by flow cytometry (surface markers), alizarin red (osteogenic capabilities), alcian blue (chondrogenic capabilities) and oil red O (adipogenic capabilities). Following culture under hypoxia conditions (1% O2) for varying time periods, the proliferation, migration and odontoblastic differentiation of HDPCs were measured using Cell Counting Kit-8, wound healing and Transwell migration assays, alkaline phosphatase staining and activity tests and western blotting (runt-related transcription factor 2, collagen I, osterix and osteopontin), respectively. Immunofluorescence and western blotting were performed to measure the expression levels of hypoxia-inducible factor-1α, pro-fission dynamin-related protein 1, mitochondria-related proteins translocase of inner mitochondrial membrane 23 and translocase of outer mitochondrial membrane 20, in addition to those of autophagy markers (p62, LC3II, Beclin-1 and autophagy-related 5). Transmission electron microscopy was also used to image the autophagosomes and mitochondrial morphology. In addition, to study the functional role of FUNDC1, its expression was silenced by liposome-mediated transfection with small interfering RNA into HDPCs. Compared with those in HDPCs cultured under normoxic conditions (21% O2), the ability of autophagy in HDPCs cultured under hypoxic conditions for 18 h was markedly increased, whilst the proliferation, migration and odontoblastic differentiation were also enhanced. Increased numbers of autophagosomes could also be observed in the hypoxic group. However, FUNDC1 knockdown in HDPCs reversed the aforementioned effects. Overall, data from the present study suggest that hypoxia can promote the proliferation, migration and odontoblastic differentiation of HDPCs, where the underlying mechanism may be associated with the activation of mitophagy downstream of FUNDC1.

Sodium Tanshinone IIA Sulfonate Improves Adverse Ventricular Remodeling Post-MI by Reducing Myocardial Necrosis, Modulating Inflammation, and Promoting Angiogenesis.

Myocardial infarction (MI) leads to pathological cardiac remodeling and heart failure. Sodium tanshinone IIA sulfonate (STS) shows to possess therapeutic potential. The present study aimed to explore the potential role of STS in ventricular remodeling post-MI. Mice were randomly divided into sham, MI + normal saline (NS) and MI + STS (20.8 mg/kg/day intraperitoneally) groups. MI was established following left anterior descending artery ligation. Cardiac function was evaluated using echocardiography. Scar size and myocardial fibrosis-associated markers were detected using Masson's trichrome staining and western blot analysis (WB). Necrosis and inflammation were assessed using H&E staining, lactate dehydrogenase (LDH) detection, ELISA, immunohistochemical staining, and WB. Furthermore, angiogenesis markers and associated proteins were detected using immunohistochemical staining and WB. Mice treated with STS exhibited significant improvements in cardiac function, smaller scar size, and low expression levels of α-smooth muscle actin and collagen I and III at 28 days following surgery, compared with the NS-treated group. Moreover, treatment with STS reduced eosinophil necrosis, the infiltration of inflammatory cells, plasma levels of LDH, high mobility group protein B1, interleukin-1β and tumor necrosis factor- α, and protein expression of these cytokines at 3 days. Macrophage infiltration was also decreased in the STS group in the early phase. Additionally, CD31+ vascular density, protein levels of hypoxia-inducible factor- 1α, and vascular endothelial growth factor were elevated in the STS-treated mice at 28 days. STS improved pathological remodeling post-MI, and the associated therapeutic effects may be a result of a decrease in myocardial necrosis, modulation of inflammation, and an increase in angiogenesis.

The effect of spironolactone on the levels of GATA4 protein and hypoxia inducible factor-1α in patients receiving coronary stent implantation

The effect of spironolactone on the levels of GATA4 protein and hypoxia inducible factor-1α in patients receiving coronary stent implantation

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

PurposePrimary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2] (2.5%; [O2]2.5) and room air ([O2]A).MethodsPrimary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2]2.5 and [O2]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates.ResultsHCEnCs grown at [O2]A and [O2]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2]2.5 than at [O2]A. HIF-1α was undetectable. Cells displayed greater viability at [O2]2.5 than at [O2]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates.ConclusionsPrimary HCEnCs show unique metabolic characteristics at physiologic [O2]. The effect of [O2] for optimization of HCEnC culture conditions should be considered.Translational RelevanceWith the advance of cell-based therapeutics for corneal endothelial diseases, [O2] should be considered an important variable in the optimization of HCEnC culture conditions.

Downregulation of topoisomerase 1 and 2 with acriflavine sensitizes bladder cancer cells to cisplatin-based chemotherapy.

Resistance to cisplatin is a major obstacle to effective treatment of bladder cancer (BC). The present study aimed to determine whether a combination of acriflavine (ACF) with cisplatin could potentiate the antitumor property of cisplatin against the BC cells. Furthermore, the molecular mechanism behind the anticancer action of ACF was considered. Two human BC cells (5637 and EJ138) contain mutated form of p53 was culture in standard condition. Cotreatment protocol (simultaneous combination of IC30 value of ACF + various dose of cisplatin for 72h) and pretreatment protocol (pretreatment with IC15 value of ACF for 24h + various dose of cisplatin for 48h) was used to determine the effect of ACF on the cells' sensitivity to main drug cisplatin. To assess the mechanism of action of ACF, real-time PCR was used to evaluate mRNA levels of hypoxia-inducible factor-1α (HIF-1α), Bax, Bcl-2, topoisomerase1 (TOP1) and topoisomerase 2 (TOP2A). Combination of ACF with cisplatin either as cotreatment or opretreatment protocol could significantly reduce the IC50 values of cisplatin as compared to the IC50 of cisplatin when use as a single drug. In addition, ACF could markedly decrease mRNA expression of TOP1 and TOP2A without changing the expression of HIF-1ɑ, Bax and Bcl-2. Our findings indicate that combination of cisplatin with ACF was able to significantly enhance the sensitivity of BC cells to cisplatin. The antitumor activity of ACF is exerted through the downregulation of TOP1 and TOP2A genes expression. ACF may serve as an adjuvant to boost cisplatin-based chemotherapy.

Calcium ion cross-linked sodium alginate hydrogels containing deferoxamine and copper nanoparticles for diabetic wound healing

Chronic non-healing diabetic wounds and ulcers can be fatal, lead to amputations, and remain a major challenge to medical, and health care sectors. Susceptibility to infection and impaired angiogenesis are two central reasons for the clinical consequences associated with chronic non-healing diabetic wounds. Herein, we successfully developed calcium ion (Ca2+) cross-linked sodium alginate (SA) hydrogels with both pro-angiogenesis and antibacterial properties. Our results demonstrated that deferoxamine (DFO) and copper nanoparticles (Cu-NPs) worked synergistically to enhance the proliferation, migration, and angiogenesis of human umbilical venous endothelial cells in vitro. Results of colony formation assay indicated Cu-NPs were effective against E. coli and S. aureus in a dose-dependent manner in vitro. An SA hydrogel containing both DFO and Cu-NPs (SA-DFO/Cu) was prepared using a Ca2+ cross-linking method. Cytotoxicity assay and colony formation assay indicated that the hydrogel exhibited beneficial biocompatible and antibacterial properties in vitro. Furthermore, SA-DFO/Cu significantly accelerated diabetic wound healing, improved angiogenesis and reduced long-lasting inflammation in a mouse model of diabetic wound. Mechanistically, DFO and Cu-NPs synergistically stimulated the levels of hypoxia-inducible factor 1α and vascular endothelial growth factor in vivo. Given the pro-angiogenesis, antibacterial and healing properties, the hydrogel possesses high potential for clinical application in refractory wounds.

Semaphorin 3A attenuates the hypoxia suppression of osteogenesis in periodontal ligament stem cells.

The occurrence and development of periodontitis are closely related to hypoxia of the periodontal microenvironment. Periodontal ligament stem cells (PDLSCs) are considered to have potential to regenerate periodontal tissues. Semaphorin 3A (Sema3A) plays an essential role in promoting osteogenesis. However, the effect of Sema3A on osteogenesis of PDLSCs under hypoxia remains unclear. The aim of this study was to investigate the effect of Sema3A on osteogenesis of PDLSCs under hypoxia. Isolated PDLSCs were identified using flow cytometry. Adipogenic differentiation potential was identified by oil red O staining. Osteogenesis was measured using Alizarin Red S staining and ALP staining. Intracellular hypoxia was induced using cobalt chloride (CoCl2 ). The expression level of hypoxia-inducible factor-1α (HIF-1α) was detected via ELISA. Expression of osteogenic markers and Sema3A was analyzed using western blot and real-time PCR. The proliferation and osteogenesis of PDLSCs were markedly inhibited with increased concentrations of CoCl2 . Under the treatment with a low concentration of CoCl2 , expression of related osteogenic markers and Sema3A decreased in a time-dependent manner. ARS and ALP staining results also showed that osteogenic calcification decreased under hypoxia. Apigenin, an inhibitor of HIF-1α, effectively up-regulated expression of Sema3A and osteogenic markers with CoCl2 treatment. Moreover, exogenous Sema3A significantly increased the expression of osteogenesis-related markers and mineralization of PDLSCs according to ALP and ARS staining with CoCl2 treatment. Hypoxia markedly inhibited osteogenesis of PDLSCs. Sema3A explicitly attenuated the hypoxia suppression of osteogenesis in PDLSCs.

Advanced platelet-rich fibrin promotes the paracrine function and proliferation of adipose-derived stem cells and contributes to micro-autologous fat transplantation by modulating HIF-1α and VEGF

BackgroundThe micro-autologous fat transplantation (MAFT) technique has demonstrated its feasibility in multiple medical fields, such as facial rejuvenation. Advanced platelet-rich fibrin (APRF), an autologous platelet concentrated on a fibrin membrane without added external factors, has shown significant potential for tissue restoration. However, the role of APRF in the modulation of MAFT remains unclear. Here, we aimed to explore the effect of APRF on MAFT.MethodsAdipose-derived stem cells (ASCs) were isolated from human gastric subcutaneous fat and treated with APRF. ELISA assays measured cytokines. The proliferation of ASCs was analyzed by CCK-8 assays. The levels of hypoxia-inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), insulin like growth factor 2 (IGF-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) were measured by ELISA assays, quantitative reverse transcription-PCR (qRT-PCR), and Western blot analysis. The effect of APRF/HIF-1α/VEGF on MAFT in vivo was analyzed in Balb/c nude mice. The BALB/c mice were subcutaneously co-transplanted with fat, APRF, and control shRNA, HIF-1α shRNA, or VEGF shRNA into the dorsal area. The serum and protein levels of the above cytokines were analyzed by ELISA assays and Western blot analysis. Lipid accumulation was measured by Oil Red O staining. The expression of CD34 was assessed by immunohistochemical staining.ResultsAPRF continuously secreted multiple cytokines, including epidermal growth factor (EGF), FGF-2, insulin like growth factor 1 (IGF-1), interleukin-1beta (IL-1β), interleukin-4 (IL-4), platelet-derived growth factor alpha polypeptide b (PDGF-AB), platelet-derived growth factor beta polypeptide b (PDGF-BB), transforming growth factor-beta (TGF-β), and VEGF. APRF was able to promote the proliferation of ASCs. APRF dose-dependently activated the expression of HIF-1α, HSP70, IGF-2, IL-6, IL-8, and VEGF in ASCs. APRF regulated the paracrine function of ASCs by modulating HIF-1α and VEGF. APRF increased the survival of MAFT by modulating HIF-1α and VEGF in vivo.ConclusionsAPRF promotes the paracrine function and proliferation of ASCs and contributes to MAFT by modulating HIF-1α and VEGF. Our findings provide new insights into the mechanism by which APRF regulates MAFT.

Neutrophil Cyto-Pharmaceuticals Suppressing Tumor Metastasis via Inhibiting Hypoxia-Inducible Factor-1α in Circulating Breast Cancer Cells.

Circulating tumor cells (CTCs) are reported as the precursor of tumor metastases, implying that stifling CTCs would be beneficial for metastasis prevention. However, challenges remain for the application of therapies that aim at CTCs due to lack of effective CTC-targeting strategy and sensitive therapeutic agents. Herein, a general CTC-intervention strategy based on neutrophil cyto-pharmaceuticals is proposed for suppressing CTC colonization and metastasis formation. Breast cancer 4T1 cells are infused as the mimic CTCs, and 4T1 cells trapped are first elucidated in neutrophil extracellular traps (NETs) expressing high levels of hypoxia-inducible factor-1α (HIF-1α) due to NET formation and thus promoting tumor cell colonization through enhanced migration, invasion and stemness. After verifying HIF-1α as a potential target for metastasis prevention, living neutrophil cyto-pharmaceuticals (CytPNEs) loaded with HIF-1α inhibitor are fabricated to therapeutically inhibit HIF-1α. It is demonstrated that CytPNEs can specially convey the HIF-1α inhibitor to 4T1 cells according to the inflammatory chemotaxis of neutrophils and down-regulate HIF-1α, thereby inhibiting metastasis and prolonging the median survival of mice bearing breast cancer lung metastasis. The research offers a new perspective for understanding the mechanism of CTC colonization, and puts forward the strategy of targeted intervention of CTCs as a meaningful treatment for tumor metastasis.

Protective Effects of Glutamine and Leucine Supplementation on Sepsis-Induced Skeletal Muscle Injuries.

This study investigated the effects of l-glutamine (Gln) and/or l-leucine (Leu) administration on sepsis-induced skeletal muscle injuries. C57BL/6J mice were subjected to cecal ligation and puncture to induce polymicrobial sepsis and then given an intraperitoneal injection of Gln, Leu, or Gln plus Leu beginning at 1 h after the operation with re-injections every 24 h. All mice were sacrificed on either day 1 or day 4 after the operation. Blood and muscles were collected for analysis of inflammation and oxidative damage-related biomolecules. Results indicated that both Gln and Leu supplementation alleviated sepsis-induced skeletal muscle damage by reducing monocyte infiltration, calpain activity, and mRNA expression levels of inflammatory cytokines and hypoxia-inducible factor-1α. Furthermore, septic mice treated with Gln had higher percentages of blood anti-inflammatory monocytes and muscle M2 macrophages, whereas Leu treatment enhanced the muscle expressions of mitochondrion-related genes. However, there were no synergistic effects when Gln and Leu were simultaneously administered. These findings suggest that both Gln and Leu had prominent abilities to attenuate inflammation and degradation of skeletal muscles in the early and/or late phases of sepsis. Moreover, Gln promoted the switch of leukocytes toward an anti-inflammatory phenotype, while Leu treatment maintained muscle bioenergetic function.

The protein tyrosine kinase inhibitor genistein suppresses hypoxia-induced atrial natriuretic peptide secretion mediated by the PI3K/Akt-HIF-1α pathway in isolated beating rat atria.

Genistein, an isoflavonoid that can inhibit protein tyrosine kinase (PTK) phosphorylation, has been shown to play pivotal roles in the signal transduction pathways of hypoxic disorders. In this study, we established a rat model of isolated beating atrium and investigated the regulator role of genistein and its downstream signaling pathways in acute hypoxia-induced atrial natriuretic peptide (ANP) secretion. Radioimmunoassay was used to detect the ANP content in the atrial perfusates. Western blot analysis was used to determine the protein level of hypoxia-inducible factor 1α (HIF-1α), and GATA4 in the atrial tissue. The results showed that acute hypoxia substantially promoted ANP secretion, whereas this effect was partly attenuated by the PTKs inhibitor genistein (3 μM). By Western blotting analysis, we found that hypoxia-induced increase in phosphorylation of Akt and transcriptional factors, including HIF-1α, were also reversed by genistein. The perfused HIF-1α inhibitors rotenone (0.5μM) or CAY10585 (10μM) plus genistein significantly abolished the enhanced ANP section induced by hypoxia. Additionally, the perfused PI3K/Akt agonist insulin-like growth factor 1 (30μM) also abolished ANP secretion induced by genistein and inhibited expression of HIF-1α. In summary, our data suggested that acute hypoxia markedly increased ANP secretion by PTKs through the phosphoinositide-3 kinase (PI3K)/HIF-1α dependent pathway.

SETD7 regulates chondrocyte differentiation and glycolysis via the Hippo signaling pathway and HIF‑1α.

Chondrocytes are well adapted to hypoxia and produce more functional extracellular matrix in low oxygen environments in vitro. In our previous study, methyltransferase SET domain containing (SETD)7 regulated chondrocyte activity in hypoxic conditions. However, the precise association between SETD7 and chondrocyte differentiation under low oxygen partial pressure remains unclear. The association between SETD7 and chondrocyte differentiation was studied by silencing SETD7 in chondrocytes in vitro. The results showed that the silencing of SETD7 in ATDC5 cells inhibited the Hippo signaling pathway, decreased Yes-associated protein (YAP) phosphorylation and increased the levels of YAP and hypoxia inducible factor-1α (HIF-1α) in the nucleus. YAP combined with HIF-1α to form a complex that promoted the expression of genes involved in chondrogenic differentiation and the glycolytic pathway. Thus, SETD7 inhibited chondrocyte differentiation and glycolysis via the Hippo signaling pathway. The present study demonstrated that SETD7 was a potential molecular target that maintained the chondrocyte phenotype during cartilage tissue engineering and cartilage-associated disease.

Vascular characteristics and expression of hypoxia genes in Tibetan pigs' hearts.

BackgroundTibetan pigs have exhibited unique characteristics from low‐altitudes pigs and adapted well to the Qinghai‐Tibet Plateau.ObjectivesThe current study was undertaken to investigate the hypoxic adaptation of heart in Tibetan pigs.MethodsThe hearts of Tibetan pigs and Landrace pigs raised at high or low altitudes were compared using 3D casting technology, scanning electron microscopy and real‐time quantitative PCR (qRT‐PCR).ResultsWe found that the ratio of the major axis to the minor axis and the density of the heart were significantly higher in Tibetan pigs than in Landrace pigs (p < 0.05). Tibetan pigs had larger diameters and higher densities of arterioles than Landrace pigs (p < 0.05), and these features have a similar variation with the expression of vascular endothelial growth factor (VEGF). The cardiac expression levels of hypoxia‐inducible factor‐1α (HIF‐1α) and endothelial nitric oxide synthase (eNOS) were significantly higher in pigs reared at high altitudes than in those reared at low altitudes (p < 0.05). In contrast, Egl nine homolog 1 (EGLN1) had the opposite trend with respect to HIF‐1α and eNOS and was related to red blood cell (RBC) counts. Notably, the expressions of erythropoietin (EPO) and endothelial PAS domain‐containing protein 1 (EPAS1) were significantly higher in Landrace pigs kept at high altitudes than in the others (p < 0.05) and were associated with haemoglobin.ConclusionsThese findings show that the regulation of the heart function of Tibetan pigs in a hypoxic environment is manifested at various levels to ensure the circulation of blood under extreme environmental conditions.

Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer.

Hypoxia is a common feature of solid tumors including stomach cancer (SC) and is closely associated with cancer malignant progression. N6-methyladenosine (m6A), a common modification on RNA, is involved in the regulation of RNA fate and hypoxic responses in cancers. However, the interaction between m6A reader insulin-like growth factor-II mRNA-binding protein 3 (IGF2BP3) and SC hypoxic microenvironment is poorly defined. In the present study, expression levels of IGF2BP3 and hypoxia inducible factor-1α (HIF1A) were examined by bioinformatics analysis and RT-qPCR and western blot assays. Cell migratory ability was assessed through Transwell and wound healing assays. The angiogenic potential was evaluated by VEGF secretion, tube formation, and chick embryo chorioallantoic membrane (CAM) assays. The interaction between IGF2BP3 and HIF1A was explored using bioinformatics analysis and RIP and luciferase reporter assays. The results showed that IGF2BP3 and HIF1A were highly expressed in SC tissues and hypoxia-treated SC cells. IGF2BP3 knockdown inhibited hypoxia-induced cell migration and angiogenesis in SC. IGF2BP3 positively regulated HIF1A expression by directly binding to a specific m6A site in the coding region of HIF1A mRNA in SC cells. HIF1A overexpression abrogated the effects of IGF2BP3 knockdown on hypoxia-induced cell migration and angiogenesis in SC. In conclusion, IGF2BP3 knockdown inhibited hypoxia-induced cell migration and angiogenesis by down-regulating HIF1A in SC.

EVT-701 is a novel selective and safe mitochondrial complex 1 inhibitor with potent anti-tumor activity in models of solid cancers.

Targeting the first protein complex of the mitochondrial electron transport chain (MC1) in cancer has become an attractive therapeutic approach in the recent years, given the metabolic vulnerabilities of cancer cells. The anticancer effect exerted by the pleiotropic drug metformin and the associated reduction in hypoxia‐inducible factor 1α (HIF‐1α) levels putatively mediated by MC1 inhibition led to the development of HIF‐1α inhibitors, such as BAY87‐2243, with a more specific MC1 targeting. However, the development of BAY87‐2243 was stopped early in phase 1 due to dose‐independent emesis and thus there is still no clinical proof of concept for the approach. Given the importance of mitochondrial metabolism during cancer progression, there is still a strong therapeutic need to develop specific and safe MC1 inhibitors. We recently reported the synthesis of compounds with a novel chemotype and potent action on HIF‐1α degradation and MC1 inhibition. We describe here the selectivity, safety profile and anti‐cancer activity in solid tumors of lead compound EVT‐701. In addition, using murine models of lung cancer and of Non‐Hodgkin's B cell lymphoma we demonstrated that EVT‐701 reduced tumor growth and lymph node invasion when used as a single agent therapy. LKB1 deficiency in lung cancer was identified as a potential indicator of accrued sensitivity to EVT‐701, allowing stratification and selection of patients in clinical trials. Altogether these results support further evaluation of EVT‐701 alone or in combination in preclinical models and eventually in patients.

Profiles of messenger RNAs and MicroRNAs in hypoxia-induced hepatic stellate cells.

BackgroundMicroRNA (miRNA) plays an important role in hepatic stellate cell (HSCs) activation and liver fibrosis. The purpose of this study is to explore the effect of hypoxia on the differential expression of mRNAs and miRNAs in rat HSCs.MethodsHSC-T6 cells were treated with cobalt chloride (CoCl2), and the activity of HSC-T6 cells was measured by the CCK-8 assay. The mRNA expression levels of hypoxia inducible factor-1α (HIF-1α), collagen type I, transforming growth factor-β1 (TGF-β1), and Smad7 were measured by RT-qPCR. The protein expression levels of HIF-1α, Bax, Bcl-2, and caspase-3 were assayed by western blot. We used basal medium and 400 µmol/L CoCl2 medium to treat HSC-T6 cells for 48 h. Cells were harvested after 48 h to extract RNA. Transcriptome sequencing was performed to investigate differentially expressed miRNAs and mRNAs (fold change >2; P<0.05). Bioinformatics analysis was performed to predict the functions of differentially expressed miRNAs and mRNAs. Further, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ResultsWith the increase of CoCl2 concentration, the activity of HSC-T6 cells decreased (P<0.05). The mRNA expression levels of HIF-1α, collagen I, TGF-β1, and Smad7, and the protein expressions levels of HIF-1α, Bax, caspase-3, and the Bcl-2/Bax ratio were increased compared with the control group (P<0.05), while the expression of Bcl-2 decreased. A total of 54 miRNAs (20 upregulated and 34 downregulated) and 1,423 mRNAs (685 upregulated and 738 downregulated) were differentially expressed in the 400 µmol/L CoCl2 medium group compared to the control basal medium group. Further bioinformatics analysis demonstrated that the differentially expressed mRNAs and miRNAs were mainly enriched in the synthesis of extracellular matrix. In addition, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ConclusionsOur results presented the profiles of mRNAs and miRNAs in hypoxia-induced HSC-T6 cells in rats, the signaling pathways, and co-expression networks. These findings may suggest novel insights for the early diagnosis and treatment of HSC activation and liver fibrosis.

Pharmacological Inhibition of MCT4 Reduces 4-Hydroxytamoxifen Sensitivity by Increasing HIF-1α Protein Expression in ER-Positive MCF-7 Breast Cancer Cells.

The rate of glycolysis in cancer cells is higher than that of normal cells owing to high energy demands, which results in the production of excess lactate. Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, play a critical role in maintaining an appropriate pH environment through lactate transport, and their high expression is associated with poor prognosis in breast cancer. Thus, we hypothesized that inhibition of MCTs is a promising therapeutic target for adjuvant breast cancer treatment. We investigated the effect of MCT inhibition in combination with 4-hydroxytamoxifen (4-OHT), an active metabolite of tamoxifen, using two estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. Lactate transport was investigated in cellular uptake studies. The cytotoxicity of 4-OHT was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In both cell lines evaluated, MCT1 and MCT4 were constitutively expressed at the mRNA and protein levels. [14C]-L-lactate uptake by both cells was significantly inhibited by bindarit, a selective MCT4 inhibitor, but weakly affected by 5-oxoploline (5-OP), a selective MCT1 inhibitor. The results of the MTT assay showed that combination with bindarit, but not 5-OP, decreased 4-OHT sensitivity. Bindarit significantly increased the levels of hypoxia-inducible factor-1α (HIF-1α) in MCF-7 cells. Moreover, HIF-1α knockdown significantly increased 4-OHT sensitivity, whereas induction of HIF-1α by hypoxia decreased 4-OHT sensitivity in MCF-7 cells. In conclusion, pharmacological MCT4 inhibition confers resistance to 4-OHT rather than sensitivity, by increasing HIF-1α protein levels. In addition, HIF-1α inhibition represents a potential therapeutic strategy for enhancing 4-OHT sensitivity.