HIF-1α Gene Expression Research Articles

Mitochondrial capacity, oxidative damage and hypoxia gene expression are associated with age-related division of labor in honey bee (Apis mellifera L.) workers.

During adult life, honey bee workers undergo a succession of behavioral states. Nurse bees perform tasks inside the nest, and when they are about 2-3 weeks old they initiate foraging. This switch is associated with alterations in diet, and with the levels of juvenile hormone and vitellogenin circulating in hemolymph. It is not clear whether this behavioral maturation involves major changes at the cellular level, such as mitochondrial activity and the redox environment in the head, thorax and abdomen. Using high-resolution respirometry, biochemical assays and RT-qPCR, we evaluated the association of these parameters with this behavioral change. We found that tissues from the head and abdomen of nurses have a higher oxidative phosphorylation capacity than those of foragers, while for the thorax we found the opposite situation. As higher mitochondrial activity tends to generate more H2O2, and H2O2 is known to stabilize HIF-1α, this would be expected to stimulate hypoxia signaling. The positive correlation that we observed between mitochondrial activity and hif-1α gene expression in abdomen and head tissue of nurses would be in line with this hypothesis. Higher expression of antioxidant enzyme genes was observed in foragers, which could explain their low levels of protein carbonylation. No alterations were seen in nitric oxide (NO) levels, suggesting that NO signaling is unlikely to be involved in behavioral maturation. We conclude that the behavioral change seen in honey bee workers is reflected in differential mitochondrial activities and redox parameters, and we consider that this can provide insights into the underlying aging process.

Effects of Dietary Astaxanthin on Growth Performance, Hepatic Antioxidative Activity, hsp70, and HIF-1α Gene Expression of Juvenile Golden Pompano (Trachinotus ovatus)

The present study was conducted to investigate the effects of different astaxanthin (AST) levels on growth performance, hepatic antioxidative activity, heat shock protein 70 (hsp70), and hypoxia-inducible factor-1 alpha (HIF-1α) gene expression of juvenile golden pompano. Fish (mean initial body weight 5.8±0.05g) were fed six isonitrogenous and isoenergetic diets (AST-0, AST-0.005, AST-0.01, AST-0.05, AST-0.1, AST-0.2) containing various supplemented levels of astaxanthin (0%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, respectively) in triplicate for 8 weeks. Growth performance (final body weight, FBW; weight gain, WG), and survival of fish fed diets containing astaxanthin above 0.01% were significantly higher (P<0.05) than of fish fed AST-0 and AST-0.005 diets; feed conversion ratio (FCR) showed the opposite trend (P<0.05). Fish fed 0.01% astaxanthin diet showed the highest value of survival. Hepatic antioxidant status (total antioxidant status, TAS; superoxide dismutase, SOD; carbonyl protein content) of shrimp improved significantly when dietary astaxanthin was over 0.01%. Relative expression profiles of hepatic hsp 70 mRNA and HIF-1α mRNA increased with increased dietary astaxanthin levels. The relative expression profiles of hepatic hsp 70 mRNA and HIF-1α mRNA of shrimp fed diets containing astaxanthin above 0.01% were obviously higher than those of shrimp fed AST-0 and AST-0.005 diets. Linear regression analysis on WG and HIF-1a mRNA indicated that the optimum dietary AST levels for optimal growth and immunity of juvenile pompano were 0.011% and 0.013%, respectively.

Effects of Dietary Astaxanthin on Growth Performance, Hepatic Antioxidative Activity, hsp70, and HIF-1α Gene Expression of Juvenile Golden Pompano (Trachinotus ovatus)

The present study was conducted to investigate the effects of different astaxanthin (AST) levels on growth performance, hepatic antioxidative activity, heat shock protein 70 (hsp70), and hypoxia-inducible factor-1 alpha (HIF-1α) gene expression of juvenile golden pompano. Fish (mean initial body weight 5.8±0.05g) were fed six isonitrogenous and isoenergetic diets (AST-0, AST-0.005, AST-0.01, AST-0.05, AST-0.1, AST-0.2) containing various supplemented levels of astaxanthin (0%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, respectively) in triplicate for 8 weeks. Growth performance (final body weight, FBW; weight gain, WG), and survival of fish fed diets containing astaxanthin above 0.01% were significantly higher (P<0.05) than of fish fed AST-0 and AST-0.005 diets; feed conversion ratio (FCR) showed the opposite trend (P<0.05). Fish fed 0.01% astaxanthin diet showed the highest value of survival. Hepatic antioxidant status (total antioxidant status, TAS; superoxide dismutase, SOD; carbonyl protein content) of shrimp improved significantly when dietary astaxanthin was over 0.01%. Relative expression profiles of hepatic hsp 70 mRNA and HIF-1α mRNA increased with increased dietary astaxanthin levels. The relative expression profiles of hepatic hsp 70 mRNA and HIF-1α mRNA of shrimp fed diets containing astaxanthin above 0.01% were obviously higher than those of shrimp fed AST-0 and AST-0.005 diets. Linear regression analysis on WG and HIF-1a mRNA indicated that the optimum dietary AST levels for optimal growth and immunity of juvenile pompano were 0.011% and 0.013%, respectively.

HIF-1α Upregulation in TP53 Disrupted Chronic Lymphocytic Leukemia Cells and Its Potential Role As a Therapeutic Target

Background: In chronic lymphocytic leukemia (CLL), disease aggressiveness and drug responsiveness can be ascribed to intrinsic genetic features of the tumor cells, such as TP53 disruption, and to interactions of CLL cells with stromal cells (SC) of the tumor microenvironment. The transcription factor HIF-1α is critically involved in the regulation of genes implicated in key cellular processes, such as cell survival and tumor progression, and also modulates the interactions of CLL cells with SC. HIF-1α expression and transcriptional activity depend on genetic alterations of tumor suppressor genes (e.g. TP53), and on extrinsic signals such as oxygen deprivation and soluble factors. In CLL cells, HIF-1α is active even in normoxia, and its expression is rapidly elevated during hypoxia. We have already reported that HIF-1α activity in CLL cells is upregulated by SC, via activation of Akt, and Ras/ERK1-2 and RhoA/RhoA kinase signaling pathways. SC are well known pro-survival factors, which protect CLL cells from spontaneous apoptosis and fludarabine-induced cytotoxicity. The cytotoxic effects of HIF-1α inhibition, and the ability of HIF-1α targeting agents to reverse constitutive or SC-induced fludarabine resistance, need to be investigated.Aim: The aim of this study was twofold: 1) to unravel the HIF-1α regulatory pathways in CLL cells, also discriminating between TP53 wild type (TP53wt)and disrupted (TP53dis), and 2) to evaluate the ability of HIF-1α inhibition to exert a direct cytotoxic effect and potentiate fludarabine cytotoxicity.Methods: CLL cells were considered TP53dis when TP53 mutation or 17p deletion (>10%) were present (n=33). Otherwise they were considered TP53wt(n=49). HIF-1α gene and protein expression were evaluated by RT-PCR and Western Blot. HIF-1α transcriptional activity was evaluated by ELISA in nuclear extracts. CLL cells were cultured alone or with the M2-10B4 SC line under normoxic or hypoxic conditions. CLL cell cultures were exposed to fludarabine (F-ara-A, 10 μM), BAY 87-2243 (BAY, 1 μM), simvastatin (1 μM) or idelalisib (10 μM), used as single agents or in combination. Cell viability was analyzed by AnnexinV/propidium Iodide immunostaining and flow cytometry. Samples were considered resistant to fludarabine when the relative viability of CLL cells exposed to F-ara-A compared to untreated controls was >0.43 (median value for the whole cohort).Results: TP53dis CLL cells expressed a constitutively higher amount of HIF-1α protein compared to TP53wt CLL cells. This upregulation was not due to a higher HIF-1α gene expression, and was associated to a significantly higher transcriptional activity of HIF-1α (p=0.009). We also evaluated the effect of extrinsic factors on the regulation of HIF-1α. We observed a further increase in HIF-1α expression when both TP53dis and TP53wt CLL cells were cultured under hypoxia. Similarly, the co-culture of CLL cells with SC further upregulated HIF-1α, via activation of Akt, Ras/ERK1-2 and RhoA/RhoA kinase signaling cascades, in both subsets. These inducing effects were particularly evident in TP53dis CLL cells. The specific inhibition of HIF-1α with BAY induced a significant reduction in viability of TP53dis and TP53wt CLL cells (p<0.001), confirming the role of HIF-1α in maintaining leukemic cells survival. We also evaluated the ability of BAY to sensitize constitutively resistant TP53dis CLL cells to F-ara-A. Results from these experiments showed a significant impairment of cell viability after dual treatment with BAY and F-ara-A compared to single agents and untreated control (mean viability: 21.8% BAY + F-ara-A, 34.1% BAY, 47.6% F-ara-A and 59.6% controls; p<0.01). In the last set of experiments, we investigated the role of HIF-1α in the microenvironment-mediated resistance to fludarabine. We found that the direct HIF-1α inhibition with BAY, and the targeting of Ras/ERK1-2 and PI3K/Akt signaling pathways using simvastatin or idelalisib were all able to counteract the protection exerted by SC toward F-ara-A-induced cell death, in both TP53dis and TP53wtCLL cells (p<0.03).Conclusions:Our data demonstrate that HIF-1α is constitutively overexpressed by TP53dis CLL cells compared to TP53wt, and is positively regulated by hypoxia and SC in both subsets. HIF-1α targeted inhibition results in high cytotoxicity of CLL cells, and is able to circumvent constitutive p53-related and SC-induced resistance to fludarabine. DisclosuresRossi:Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Massaia:Janssen: Other: advisory board; Roche: Other: advisory board, research support; Gilead: Other: advisory board. Boccadoro:Mundipharma: Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Coscia:Gilead: Honoraria; Janssen: Honoraria; ROCHE: Honoraria, Other: Advisory board; Mundipharma: Honoraria; Karyopharm: Research Funding.

Isoflurane neurotoxicity involves activation of hypoxia inducible factor-1α via intracellular calcium in neonatal rodents

ObjectivePreviously, we found that the inhaled anesthetic isoflurane up-regulated the transcriptional factor hypoxia inducible factor (HIF)-1α protein levels during induction of neurodegeneration in the brain of neonatal rats. Here, we investigated the role of HIF-1α and the underlying signaling pathway in the neurodegenration induced by isoflurane in rodent developing brain. MethodsPrimary hippocampal neurons were exposed to isoflurane (0.4mM) for 12h. Neuron injury was analyzed by 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) test and quantification of lactate dehydrogenaserelease. HIF-1α gene expression and transcriptional activity, cleaved caspase-3 and phosphoinositide phospholipase C (PLC)-γ gene expression were quantified. Cytosolic calcium concentration was measured by calcium imagining. The role of HIF-1α on juvenile learning and memory impairment induced by isoflurane was studied by fear conditioning and extinction test and Morris water maze test. ResultsIsoflurane induced HIF-1α gene expression and transcriptional activity in vitro. Furthermore, pharmacological inhibition of HIF-1α or knockdown of HIF-1α by shRNA counteracted the neurotoxicity of isoflurane. Ca2+ signaling pathways involving PLC-γ activation are required for isoflurane-evoked HIF-1α accumulation. Finally, partial deficiency of HIF-1α attenuated isoflurane-induced neurodegeneration in developing brain, and alleviated juvenile learning and memory impairment induced by isoflurane. ConclusionHIF-1α activation via cytosolic Ca2+ signaling pathway play a role in the mechanism of isoflurane-induced neurodegeneration in neonatal rodents, suggesting HIF-1α as a potential therapeutic target for the prevention of the deleterious effects of prolonged exposures to anesthetics.

LY6E: a conductor of malignant tumor growth through modulation of the PTEN/PI3K/Akt/HIF-1 axis.

Lymphocyte antigen 6 complex, locus E (LY6E) has been implicated in the malignant progression of various types of cancers; however, the underlying mechanism remains unclear. Here, we identified LY6E as an activator of HIF-1 and revealed their mechanistic and functional links in malignant tumor growth. The aberrant overexpression of LY6E increased HIF-1α gene expression principally at the transcription level. This, in turn, led to the expression of the pro-angiogenic factors, VEGFA and PDGFB, through decreases in the expression levels of PTEN mRNA and subsequent activation of the PI3K/Akt pathway. The LY6E-HIF-1 axis functioned to increase tumor blood vessel density and promoted tumor growth in immunodeficient mice. LY6E expression levels were significantly higher in human breast cancers than in normal breast tissues, and were strongly associated with the poor prognoses of various cancer patients. Our results characterized LY6E as a novel conductor of tumor growth through its modulation of the PTEN/PI3K/Akt/HIF-1 axis and demonstrated the validity of targeting this pathway for cancer therapy.

Analysis of BNIP3 expression and clinical research in salivary adenoid cystic carcinoma

This study investigated the expression of BNIP3 in salivary adenoid cystic carcinoma (SACC) and its correlations to the clinicopathological features and prognosis of patients with SACC. The role of BNIP3 in the progress of hypoxia-induced autophagy was elucidated. The expression levels of BNIP3, hypoxia inducible factor (HIF)-1α, and LC3 in 65 SACC cases were detected by immunohistochemical staining method, and the correlation between the expression of BNIP3 and the clinicopathological features in SACC was analyzed. In addition, the correlations of BNIP3 gene expression with HIF-1α and LC3 gene expression were analyzed. The survival rate of patients with SACC was evaluated by univa-riate survival analysis. BNIP3 was considerably expressed in SACC in all three histological patterns, and was positive in 41 cases (63.1%). BNIP3 gene expression was significantly correlated with histological grade (P=0.001) and HIF-1α gene expression (P=0.011). By contrast, BNIP3 gene expression was not significantly correlated with LC3 gene expression (P= 0.167). The overall survival rate of patients with negative BNIP3 expression was better than that of patients with positive BNIP3 expression (P<0.05). BNIP3 might play a vital role in the tumorigenesis of SACC and may be a new target for gene therapy. .

Metformin increases PDH and suppresses HIF-1α under hypoxic conditions and induces cell death in oral squamous cell carcinoma

BackgroundMetformin is a biguanide, belonging to the oral hypoglycemic agents and is a widely used in the treatment of type 2 diabetes. Evidence indicate that Metformin inhibits cell proliferation in several human cancers and inhibits the Warburg phenomenon in tumor cells.ResultsLow PDH levels were observed in OSCC, and Metformin promotes an increase in PDH levels in hypoxic conditions. Metformin also reduced HIF-1α mRNA and protein levels. Metformin demonstrated antiproliferative effects, inhibited migration, increased the number of apoptotic cells and increased the transcription of caspase 3.ObjectiveThe present study aims to explore the effects of Metformin in hypoxic conditions. Specifically, we focused on pyruvate dehydrogenase (PDH), (hypoxia-inducible factor 1α) HIF-1α levels and the oral squamous cell carcinoma (OSCC) cell phenotype. Additionally, we also investigated a theoretical consequence of Metformin treatment.MethodsPDH levels in patients with OSCC and oral dysplasia were evaluated. Metformin was administered in vitro to test the effect of Metformin under hypoxic conditions. The results were complemented by Bioinformatics analyses.ConclusionsIn conclusion, our current findings show that Metformin reduces HIF-1α gene expression and increases PDH expression. Metformin inhibits cell proliferation and migration in the OSCC cell line model. Additionally, Metformin enhances the number of apoptotic cells and caspase 3 levels. Interestingly enough, Metformin did not increase the mutant p53 levels under hypoxic conditions.

Abstract 2064: Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma

Abstract Background: Targeted therapy including VEGF and mTOR pathway inhibitors has dramatically transformed treatment options and outcomes for patients with metastatic clear cell renal cell carcinoma (ccRCC). However, alternate treatments are needed as resistance to these initially promising agents occurs frequently. RNAi interference (RNAi), an innate gene silencing mechanism, has been explored as a new class of therapeutics where conventional treatments are lacking or have failed. The challenge in leveraging this promising approach has been efficient delivery of an RNAi trigger (siRNA) to target tissue. Over 90% of ccRCC tumors express a mutant inactive form of the von Hippel-Landau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong evidence supports the observation that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF-2α), a tumorigenic driver of ccRCC. Methods: We have developed a targeted delivery platform called Dynamic Polyconjugte™ (DPC) as an RNAi-based therapeutic targeting HIF-2α for advanced ccRCC. The ccRCC-specific DPC (ITG-DPC) comprises a membrane active polymer to promote RNAi trigger endosomal release, a ligand that binds to αV-containing integrin receptors expressed on tumor cells, reversible masking to prevent polymer activity before reaching the endosomal compartment, and a potent and specific RNAi trigger to HIF-2α. The modular nature of this delivery platform allows for flexibility to optimize each functional component independently. The ligand-dependent delivery of ITG-DPC was first evaluated in cultured tumor cells and then confirmed in ccRCC tumors established in nude mice using fluorescently-labeled ITG-DPC and confocal microscopy. To validate silencing of HIF-2α as an effective therapeutic approach, an inducible shRNA to HIF-2α was expressed in ccRCC tumors established in mice that significantly silenced HIF-2α gene expression and induced tumor regression. Results: Bi-weekly injection of ITG-DPC into nude mice with established orthotopic A498 ccRCC tumors resulted in &amp;gt;80% knockdown of HIF-2α mRNA and significant tumor growth inhibition. Histological examination of tumor sections showed substantial cell killing and destruction of tumor architecture. Down-regulation of HIF-2α regulated pathways genes including VEGF-A, and the corresponding reduction in tumor-associated CD31 positive neovascularization, corroborated on-target effects of HIF-2α gene silencing. Conclusion: Targeted delivery of a HIF-2α specific RNAi-based-therapeutic has the potential to radically impact the late-stage ccRCC treatment paradigm. Citation Format: So Wong, Weijun Cheng, Darren Wakefield, Aaron Almeida, Andrei Blokhin, Holly Hamilton, Vladimir Subbotin, Julia Hegge, Zane Neal, Guofeng Zhang, David Rozema, David Lewis, Steven Kanner. Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2064.

Angiogenic factors are increased in circulating granulocytes and CD34+ cells of myeloproliferative neoplasms.

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF-1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF-1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34+ cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFβ and MAPK signaling pathways were detected in CD34+ cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation-related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors. © 2016 Wiley Periodicals, Inc.

SNAIL gene inhibited by hypoxia-inducible factor 1α (HIF-1α) in epithelial ovarian cancer.

The aim of this study was to investigate the relationship between HIF-1α and SNAIL gene expression in the epithelial ovarian cancer (EOC) cell line. EOC cells were treated with hypoxia, hypoxia combined with rapamycin, and control. The expression of HIF-1α and E-cad were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The gene expression of SNAIL was studied by RT-PCR and real-time PCR. RNA interference technology was used to determine the relationship between HIF-1α and SNAIL. The present study indicated that the HIF-1α protein was expressed and increased in EOC cell line. SNAIL mRNA was found to increase and E-cad expression decreased with the time of hypoxia prolonged. Hypoxia increased invasion abilities of EOC cell line, but compared with cells exposed to hypoxia, the change of invasive ability of cells with rapamycin had no effect. The expression of HIF-1α protein and SNAIL mRNA could be inhibited gradually by rapamycin. siRNA of HIF-1α could suppress the expression of SNAIL while siRNA of SNAIL had no influence on HIF-1α protein expression. HIF-1α may be the upstream of the SNAIL gene in EOC. Our data suggested that HIF-1α might be an upregulator of the SNAIL gene and HIF-1α-SNAIL-E-cad pathway may play an important role in EOC invasion and metastasis.

Possible Hypoxia Signaling Induced Alteration of Glucose Homeostasis in Rats Exposed to Chronic Intermittent Hypoxia - Role of Antioxidant (Vitamin C) and Ca2+ Channel Blocker (Cilnidipine)

Background: Hyperglycemia is found to be a regulator of HIF-1α gene expression but the regulation of HIF-1α on glucose homeostasis is unclear. Objective: To determine whether chronic intermittent hypoxia (CIH) alters glucose regulation and such alterations can revert through treatments with either antioxidant (vitamin c) or calcium channel blocker (cilnidipine) in male albino rats. Methods: The rats were divided into six groups i.e. normoxia (21% oxygen), CIH (10% oxygen with cycle time 3:1.5; 8h/day), normoxia with vitamin c (50 mg /100g. b.wt, orally), CIH with vitamin c, normoxia with cilnidipine (1 mg/kg/day; ip) and CIH with cilnidipine. Serum MDA, HIF-1α, fasting plasma glucose, insulin, GTT, HOMA-IR and insulinogenic index were evaluated. Results: Serum HIF-1α and MDA concentration in rats exposed to CIH increased significantly whereas simultaneous CIH with vitamin c and CIH with cilnidipine treatment show reversion of both serum HIF- 1α and MDA concentrations towards normoxic status. CIH rats showed increased fasting glucose level with unchanged plasma insulin level but both vitamin c and cilnidipine treatment improved the status. Elevated HOMA-IR and insulinogenic index along with impaired GTT were found in CIH groups although vitamin c and cilnidipine improved the glucose homeostasis in CIH exposed rats. Conclusion: CIH induces over production of reactive oxygen species as well as hyper activities of sympathetic N-type Ca2+ channels possibly through HIF 1-α expression and influence on insulin signaling by causing hyperglycemia, glucose intolerance and insulin resistance in rats. Simultaneous treatment with vitamin c or cilnidipine improves glucose homeostasis in CIH exposed rats.

Cinnamon extract reduces VEGF expression via suppressing HIF-1α gene expression and inhibits tumor growth in mice.

Although many anti-VEGF agents are available for cancer treatment, side effects of these agents limit their application for cancer treatment and prevention. Here we studied the potential use of a diet-based agent as an inhibitor for VEGF production. Using a VEGF reporter assay, our data showed that an extract from cinnamon (CE) was a potent inhibitor of VEGF production in human cancer cells and suggested inhibition might be mediated through the suppression of HIF-1α gene expression and protein synthesis. Furthermore, CE treatment was found to inhibit expression and phosphorylation of STAT3 and AKT, which are key factors in the regulation of HIF-1α expression, and significantly reduce angiogenesis potential of cancer cells by migration assay. Consistent with these results, we observed significant suppression of VEGF expression, blood vessel formation, and tumor growth in a human ovarian tumor model in mice treated with CE. Cinnamaldehyde, a major component in cinnamon, was identified as one active component in CE that inhibits VEGF expression. Taken together, our findings provide a novel mechanism underlying anti-angiogenic and anti-tumor actions of CE and support the potential use of CE in cancer prevention and treatment. © 2016 Wiley Periodicals, Inc.

Opposite prognostic roles of HIF1α and HIF2α expressions in bone metastatic clear cell renal cell cancer.

BACKGROUNDPrognostic markers of bone metastatic clear cell renal cell cancer (ccRCC) are poorly established. We tested prognostic value of HIF1α/HIF2α and their selected target genes in primary tumors and corresponding bone metastases.RESULTSExpression of HIF2α was lower in mRCC both at mRNA and protein levels (p/mRNA/=0.011, p/protein/=0.001) while HIF1α was similar to nmRCC. At the protein level, CAIX, GAPDH and GLUT1 were increased in mRCC. In all primary RCCs, low HIF2α and high HIF1α as well as CAIX, GAPDH and GLUT1 expressions correlated with adverse prognosis, while VEGFR2 and EPOR gene expressions were associated with favorable prognosis. Multivariate analysis confirmed high HIF2α protein expression as an independent risk factor. Prognostic validation of HIFs, LDH, EPOR and VEGFR2 in RNA-Seq data confirmed higher HIF1α gene expression in primary RCC as an adverse (p=0.07), whereas higher HIF2α and VEGFR2 expressions as favorable prognostic factors. HIF1α/HIF2α-index (HIF-index) proved to be an independent prognostic factor in both the discovery and the TCGA cohort.PATIENTS AND METHODSExpressions of HIF1α and HIF2α as well as their 7 target genes were analysed on the mRNA and protein level in 59 non-metastatic ccRCCs (nmRCC), 40 bone metastatic primary ccRCCs (mRCC) and 55 corresponding bone metastases. Results were validated in 399 ccRCCs from the TCGA project.CONCLUSIONSWe identified HIF2α protein as an independent marker of the metastatic potential of ccRCC, however, unlike HIF1α, increased HIF2α expression is a favorable prognostic factor. The HIF-index incorporated these two markers into a strong prognostic biomarker of ccRCC.

Ginsenoside Rg1 and platelet-rich fibrin enhance human breast adipose-derived stem cell function for soft tissue regeneration.

Adipose-derived stem cells (ASCs) can be used to repair soft tissue defects, wounds, burns, and scars and to regenerate various damaged tissues. The cell differentiation capacity of ASCs is crucial for engineered adipose tissue regeneration in reconstructive and plastic surgery. We previously reported that ginsenoside Rg1 (G-Rg1 or Rg1) promotes proliferation and differentiation of ASCs in vitro and in vivio. Here we show that both G-Rg1 and platelet-rich fibrin (PRF) improve the proliferation, differentiation, and soft tissue regeneration capacity of human breast adipose-derived stem cells (HBASCs) on collagen type I sponge scaffolds in vitro and in vivo. Three months after transplantation, tissue wet weight, adipocyte number, intracellular lipid, microvessel density, and gene and protein expression of VEGF, HIF-1α, and PPARγ were higher in both G-Rg1- and PRF-treated HBASCs than in control grafts. More extensive new adipose tissue formation was evident after treatment with G-Rg1 or PRF. In summary, G-Rg1 and/or PRF co-administration improves the function of HBASCs for soft tissue regeneration engineering.

Beclin-1 and hypoxia-inducible factor-1α genes expression: Potential biomarkers in acute leukemia patients.

Beclin-1, an important autophagic gene, and hypoxia-inducible factor-1α (HIF-1α), the master regulator of the hypoxic response, are reported in several human cancers. However, their expressions in acute leukemia haven't yet been well investigated. This study was designed to investigate the gene expression of beclin-1, microtubule-associated protein-1 light chain-3B (MAB1LC3B), the anti-apoptotic marker Bcl-2, and HIF-1α, as well as to evaluate the relationship between their expressions profile and prognosis in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) adult patients. The study involved 30 AML patients, 25 ALL patients, and 20 controls. Gene expression was analyzed using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). In both AML and ALL groups, beclin-1 and MAB1LC3B expressions were significantly down-regulated (p < 0.001), while HIF-1α (p < 0.01) and Bcl-2 (p < 0.001) expressions were significantly up-regulated compared to the control group. HIF-1α fold expression was significantly negatively correlated with beclin-1 (p < 0.01). Moreover, decreased beclin-1 gene expression and increased HIF-1α gene expression were both associated with poor survival, supporting their pivotal role in the development and progression of acute leukemia. Both Beclin-1 and HIF-1α could be considered as important biomarkers determinants of pathogenesis and survival in acute leukemia.

Effects of β-glucan on ROS production and energy metabolism in yellow croaker (Pseudosciaena crocea) under acute hypoxic stress.

The aim of the present study was to evaluate the effect of β-glucan on acute hypoxia-induced oxidative stress and the changes in energy metabolism by determining ROS production, activities and mRNA levels of energy metabolism enzyme (PK, F-ATPase, SDH and MDH), and in gene expression of HIF-1α in the liver of large yellow croaker. Fish were injected with β-glucan at a dose of 0 or 5mgkg(-1) body weight on 6, 4 and 2days before exposed to 1.5 and 7.0 mg DO L(-1) for 48h. The results showed that β-glucan enhanced survival rate and reduced ROS during the lethal hypoxic stress, indicating that β-glucan could ameliorate hypoxia-induced oxidative stress. Obtained results also showed that β-glucan could up-regulate activities and mRNA levels of PK, demonstrating that β-glucan increased anaerobic glycolysis capacity. Furthermore, a coordinated transcriptional regulation of energy metabolism enzyme genes was observed, suggesting that HIF-1α is required for regulating these genes. In conclusion, β-glucan could alleviate cute hypoxia-induced oxidative stress in large yellow croker by enhancing anaerobic glycolysis capacity, emphasizing a central role of transcription factor HIF-1α in the process.

Acute hypoxia up-regulates HIF-1α and VEGF mRNA levels in Amazon hypoxia-tolerant Oscar (Astronotus ocellatus).

Amazon fish maintain oxygen uptake through a variety of strategies considered evolutionary and adaptive responses to the low water oxygen saturation, commonly found in Amazon waters. Oscar (Astronotus ocellatus) is among the most hypoxia-tolerant fish in Amazon, considering its intriguing anaerobic capacity and ability to depress oxidative metabolism. Previous studies in hypoxia-tolerant and non-tolerant fish have shown that hypoxia-inducible factor-1α (HIF-1α) gene expression is positively regulated during low oxygen exposure, affecting vascular endothelial growth factor (VEGF) transcription and fish development or tolerance in different manners. However, whether similar isoforms exists in tolerant Amazon fish and whether they are affected similarly to others physiological responses to improve hypoxia tolerance remain unknown. Here we evaluate the hepatic HIF-1α and VEGF mRNA levels after 3h of acute hypoxia exposure (0.5mgO2/l) and 3h of post-hypoxia recovery. Additionally, hematological parameters and oxidative enzyme activities of citrate synthase (CS) and malate dehydrogenase (MDH) were analyzed in muscle and liver tissues. Overall, three sets of responses were detected: (1) as expected, hematocrit, hemoglobin concentration, red blood cells, and blood glucose increased, improving oxygen carrying capacity and glycolysis potential; (2) oxidative enzymes from liver decreased, corroborating the tendency to a widespread metabolic suppression; and (3) HIF-1α and VEGF increased mRNA levels in liver, revealing their role in the oxygen homeostasis through, respectively, activation of target genes and vascularization. This is the first study to investigate a hypoxia-related transcription factor in a representative Amazon hypoxia-tolerant fish and suggests that HIF-1α and VEGF mRNA regulation have an important role in enhancing hypoxia tolerance in extreme tolerant species.

Adipose Tissue Hypoxia, Inflammation, and Fibrosis in Obese Insulin-Sensitive and Obese Insulin-Resistant Subjects.

A substantial number of obese individuals are relatively insulin sensitive and the etiology for this variation remains unknown. The primary objective was to detect factors in adipose tissue differentiating obese insulin-sensitive (OBIS) from obese insulin-resistant (OBIR) individuals and investigate whether adipose tissue hypoxia is a contributing factor in the pathogenesis of insulin resistance. This was a cross-sectional study in the general community. Subjects consisted of nondiabetic OBIS and OBIR subjects with similar body mass index, age, and total body fat but different insulin sensitivity index as well as lean insulin-sensitive subjects. There were no interventions. We examined adipocytokines and the expression of candidate genes regulating hypoxia, inflammation, and lipogenesis in adipose tissue and adipose tissue oxygenation. OBIS subjects had increased plasma adiponectin but similar plasma TNFα and leptin levels as compared with OBIR subjects. Genes regulating inflammation (CD68, MCP1, scavenger receptor A, and oxidized LDL receptor 1) were increased by 40%–60% (P < .05) in OBIR vs OBIS cohorts. In addition, genes involved in extracellular matrix formation such as collagen VI and MMP7 were up-regulated by 43% and 78% (P < .05), respectively, in OBIR vs OBIS. The expression of HIF1α and VEGF gene expression was increased by 37% and 52%, respectively, in OBIR vs OBIS (P < .01). Despite the differential expression in hypoxia-related genes, adipose tissue oxygenation measured by a Licox oxygen probe was not different between OBIS and OBIR subjects, but it was higher in lean subjects as compared with obese subjects. We confirmed that adipose tissue inflammation and fibrosis play an important role in the pathogenesis of insulin resistance independent of obesity in humans. Whether hypoxia is simply a consequence of adipose tissue expansion or is related to the pathogenesis of obesity-induced insulin resistance is yet to be understood.

Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines.

Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p < .05). This treatment also decreased both gene and protein expression of HIF-1α and VEGF-A under hypoxic conditions (p < .05). Among the proteins evaluated by protein array, melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p < .05). Taken together, these results showed that melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions.