HIF-1alpha Expression Research Articles

The regulation of Hypoxia-Inducible Factor-1 (HIF-1alpha) expression by Protein Disulfide Isomerase (PDI).

Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1–245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state.

L-Carnitine ameliorates the liver by regulating alpha-SMA, iNOS, HSP90, HIF-1alpha, and RIP1 expressions of CCL4-toxic rats.

Objective(s):Carbon tetrachloride (CCL4) toxicity triggers fibrosis, activating various mechanisms within the cell. We aimed to create damage with CCL4 and investigate the effectiveness of L-carnitine on the mechanisms we identified.Materials and Methods:Forty rats were divided into 5 groups with equal number of rats in each group. Group I: Control group, Group II: L-carnitine group, 200 mg/kg L-carnitine twice a week, Group III: CCL4 group, 0.2 ml/100 gr CCL4, IP, dissolved in olive oil 2 times a week during 6 weeks; Group IV: L-carnitine + CCL4 group, 200 mg/kg L-carnitine 24 hr before 0.2 ml/100 g CCL4 application twice a week; Group V: CCL4 + L-carnitine, 200 mg/kg L-carnitine half an hour after 0.2 ml/100 g CCL4 application. The liver was evaluated histologically. Immunohistochemically stained with α-SMA, iNOS, HSP90, HIF-1α, and RIP1. TNF-α, TGF-β, AST, ALT, ALP, and GGT measurements were evaluated. Results:In the classical lobule periphery, an increase in lipid accumulation and a decrease in glycogen accumulation were observed. After immunohistochemical measurements and biochemical analyzes, an increase in the expression density of all proteins was observed in group III. In group IV and V, an improvement in tissue and a decrease in protein expression densities were observed.Conclusion:iNOS serves as a free radical scavenger in response to damage caused by increased toxicity of α-SMA, HSP90, and HIF-1α. Especially, increased RIP1 level in the tissue indicates the presence of necrosis in the tissue after CCL4-toxicity. Supplementing the amount of endogenous L-carnitine with supplementation provides a significant improvement in the tissue.

The effect of topical administration of simvastatin on entochondrostosis and intramembranous ossification: An animal experiment.

BackgroundSimvastatin, a drug for lowering serum cholesterol, has been shown to enhance bone regeneration, but few studies have qualitatively and quantitatively tested its effect when used topically in different animal models. This study aims to investigate topical administration of simvastatin as a bone regeneration inducer by testing its effect on bone formation in both long tubular bone and flat bone defect, and the mechanism involved.MethodsTwo animal models were used for testing the effect of simvastatin on entochondrostosis and intramembranous ossification respectively. Simvastatin of different dosages combined with poly lactic acid were implanted in extreme radial defects of 12 adult male New Zealand rabbits. Bone formation was monitored using x-ray and CT-scan and measured using x-ray scales, pixel values and spiral CT-scan for 16 weeks before being subject to histological and immunohistochemistry examination. The result was compared with that of autograft and blank control groups. Simvastatin with thrombin and fibrin sealant were implanted in calvarial defects of three Rhesus monkeys and monitored for 18 weeks. Bone formation was compared between the simvastatin and the blank control group using spiral CT-scan and histological examination.ResultsBoth visual and quantitative measurements by x-ray and spiral CT-scan indicated significant bone formation in radial defects in all simvastatin groups and the autograft group whereas no bone formation was found in control groups. There was no significant difference in bone formation quantity between 100 ​mg simvastatin and autograft. Histological and immunohistochemistry examination indicated entochondrostosis in association with positive expression of BMP-2 and HIF-1 alpha. Spiral CT-scan and histological examination of calvarial defects of monkeys showed intramembranous ossification after simvastatin implantation. No change was found in the control group.ConclusionsTopical administration of simvastatin induces entochondrostosis and intramembranous ossification by enhancing expression of BMP-2 and HIF-1 alpha. The effect of simvastatin on bone regeneration is comparable to autograft.The translational potential of this articleTopical administration of simvastatin can repair bone defect in both long tubular bones and flat bones of rabbits and monkeys as effectively as autograft. Given that it is cheap, safe and already in clinical use, simvastatin might be considered as a bone regeneration inducer with great potential.

Endogenous reactive oxygen species and nitric oxide have opposite roles in regulating HIF-1alpha expression in hypoxic astrocytes.

Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.

Abstract 16335: Metabolic Reprogramming From Glycolysis to Amino Acid Utilization in Cardiac Hif1 Alpha Deficient Mice

Introduction: Hypoxia is an important environmental cue implicated in several physiopathological processes, including cardiac development. Several gain of function models described before indicate that HIF1 signaling needs to be tightly regulated to ensure proper heart formation. However, there is lack of consensus about the functional outcomes of cardiac HIF1 elimination. We have previously reported that HIF1alpha expression is spatiotemporally regulated along cardiogenesis, establishing metabolic territories in the embryonic myocardium and controlling a switch from glycolysis to fatty acid oxidation (FAO) essential for chamber formation and cardiomyocyte maturation. Objectives and Hypothesis: We aim to assess the consequences of cardiac deletion of HIF1alpha during heart development and identify the adaptations to HIF1 signaling loss. Based on the tight regulation of HIF1alpha expression during cardiogenesis, we anticipated significant alterations of cardiac metabolism as well as functional and structural defects in HIF1alpha mutants. Methods and Results: A new conditional Hif1alpha knock out was generated in NKX2.5 cardiac progenitors. By means of histology, electron microscopy and high-throughput genomics, proteomics and metabolomics, we found that deletion of Hif1alpha leads to impaired embryonic glycolysis without influencing cardiomyocyte size or proliferation and results in increased mitochondrial number, transient activation of amino acid response and upregulation of HIF2alpha and ATF4. HIF1alpha mutants display normal FAO metabolic profile and do not show cardiac dysfunction in the adulthood. Conclusions: We demonstrated that HIF1 signaling is dispensable for heart development and uncovered the metabolic flexibility of the mammalian embryonic myocardium, able to utilize alternative fuels to carbohydrates in contrast to other vertebrates like zebrafish. This data highlights the importance of HIF in cardiac metabolic programing and could explain the distinct proliferative and regenerative capacity of cardiomyocytes from different species in response to cardiac injury.

Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†.

Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus–oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

HYPOXIC PRECONDITIONING AMELIORATES ISCHEMIA-INDUCED DAMAGE OF ISOLATED RAT ISLETS

Aim: Islet isolation results in disruption of the native islet vascular network until posttransplant islet revascularisation is completed. Prolonged exposure to a hypoxic and nutrient-depleted environment has detrimental effects on morphological and functional integrity of isolated islets. In solid organ transplantation it was shown that short sublethal periods of anoxia can induce adaptive mechanisms in the affected tissue offering protection against subsequent potentially lethal hypoxic insults. This study was aimed to explore the potential of hypoxic preconditioning for isolated islets. Methods: Rat islets, isolated from 20 non-heartbeating donors, were subjected either to 2 x 2 min-periods of anoxia, intermitted by a 2 min-period of oxygen supply, or to sham-treatment. Hypoxic preconditioning and sham-treatment were followed by 4 hours of recovery in normoxia prior to 24 hr-culture at 1.5% oxygen. Islet characterisation included yield of islet equivalents (IEQ) or islet particle number (IN), viability (FDA-PI), DNA (pico-green) and ATP (luciferase assay) content, intraislet reactive oxygen species (ROS) production (DCFH-DA), gene expression (qRT-PCR), and glucose stimulation index (2 vs 20 vs 2 mM). Data were normalised to islet DNA content and are presented as mean ± SEM. Results: Hypoxic preconditioning had no effect on islet survival when compared with sham-treatment (64 ± 7 vs 61 ± 7%). Compared with preconditioned islets, sham-treated controls were characterised by increased fragmentation when determined as IN/IEQ ratio (1.51 ± 0.18 vs 1.24 ± 0.13, p<0.05). Fragmentation corrrelated inversely with viability of sham-treated and preconditioned islets (61 ± 3 vs 66 ± 3 %, p<0.01). ATP (0.66 ± 0.14 vs 0.73 ± 0.20 pM/ng DNA) and insulin content (51.1 ± 12.5 vs 46.3 ± 11.7 ng/ng) were not altered by hypoxic preconditioning compared with sham-treatment. ROS production was reduced after hypoxic preconditioning compared with sham-treatment (645 ± 73 vs 858 ± 88 AU/ng, p<0.01). Islets of both experimental groups responded with insulin release after glucose challenge, but the stimulation index of preconditioned islets was significantly larger compared with sham-treated islets (2.39 ± 0.37 vs 2.09 ± 0.33, p<0.05). Whilst pro-apoptotic BAX and CHOP gene expression was reduced by 54 ± 15% (p<0.05) and 49 ± 8% (p<0.05), hypoxic preconditioning increased HIF-1 alpha and VEGF-A gene expression by 90 ± 41% (p<0.01) and 184 ± 65% (p<0.05), respectively. Conclusion: Although hypoxic preconditioning could not ameliorate ischaemia-induced loss of islets, islet morphology and function was significantly improved presumably as result of reduced intraislet ROS production. qRT-PCR-determined gene expression suggests that hypoxic preconditioning induces rescue mechanisms islets and mediates a significant anti-apoptotic effect. This study describes a new option for pretransplant treatment of isolated human islets. European Union’s Cooperation Programme FP7 (HEALTH-F2-2012-305746). Oxford NIHR Biomedical Research Centre.

L-carnitine effects in CCl4-nephrotoxicity: Immunohistochemical evaluation of glomerular nephrin and HIF-1alpha expressions

Purpose: In this study, we aimed to demonstrate the effects of L-carnitine after carbontetrachloride (CCl4) toxicity through nephrin and Hypoxia inducible factor-1 alpha (HIF-1α) expressions in the glomerular structure.Materials and Methods: Forty male Sprague dawley rats were divided into 5 groups with animals in each group. Group I: Control group; 0.2 ml olive oil intraperitoneal (ip) twice weekly, Group II: L-carnitine group; 200 mg/kg L-carnitine (ip) twice a week, Group III: CCl4 group; 0.2 ml CCl4 (ip) twice a week for 6 weeks, Group IV: L-carnitine + CCl4 group, 200 mg/kg ip L-carnitine 24 hours before CCl4 twice a week, Group V: CCl4 + L-carnitine group; 200 mg/kg L-carnitine half an hour after CCl4 twice a week. Immunohistochemical staining was performed on kidney tissue sections to show nephrin and HIF-1α expression. Expression densities of the proteins were measured by ImageJ program.Results: Nephrin expression was significantly increased in Group III compared to other groups. There was a significant increase in HIF-1α expression only between Group I and Group III. Expression densities of proteins in L-carnitine-treated groups were similar to control.Conclusion: L-carnitine has both protective and therapeutic effects against CCl4 toxicity in renal glomeruli.

Abstract B29: Modeling the oxygen-deprived breast cancer tumor microenvironment within a three-dimensional bioengineered platform that exhibits hypoxia-driven immune evasion

Abstract Introduction: Oxygen deprivation within tumors has been associated with enhanced cancer cell survival, altered extracellular matrix (ECM) production, and increased immune evasion in breast cancer (BCa). These outcomes collectively contribute to chemotherapeutic challenges. Current models do not adequately mimic physiologic oxygen levels relevant to breast tissue and tumor-immune interactions. Taking this into consideration, we developed a plasma-derived three-dimensional (3D) model that supports growth of BCa cells, generates physio- and pathophysiologic oxygen levels, and supports prevailing hypoxia-driven tumor outcomes such as immune evasion. Methods: BCa cells (MCF7 and MDA-MB-231) were embedded into 3D scaffolds formed through the cross-linking of patient-derived plasma. The oxygen content within these 3D matrices was engineered and further characterized to recapitulate physiologic blood and breast tissue (3D physiologic, gradient 14-10 kPa) or pathophysiologic (3D tumorous, gradient 9.6-2.4 kPa) levels by the manipulation of height, culture time, oxygen incubation environment, and cellular concentration. BCa cells grown within 3D physiologic and tumorous scaffolds were analyzed by flow cytometry, confocal imaging, or immunohistochemistry for the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), proliferation capabilities, and ECM production. Peripheral blood mononuclear cells were incorporated on the top of 3D physiologic and tumorous scaffolds, and the differences in the tumor-infiltrating capabilities of T cells into 3D physiologic and tumorous scaffolds were assessed by flow cytometry. Lastly, hypoxic inhibition was validated as a target to sensitize BCa cells in order to overcome immune surveillance. Results: BCa cells grown in oxygen-deprived 3D tumorous environments showed a significant increase in the expression of HIF-1 alpha and a reduced cell proliferation compared to the BCa cells in 3D physiologic scaffolds. Increased secretion of collagen I, collagen III, laminin, and fibronectin was demonstrated by BCa cells in 3D tumorous compared to 3D physiologic scaffolds. T-cell infiltration (especially CD8 T cells) was significantly impaired within the oxygen-deprived breast cancer tumor microenvironment (TME), and hypoxia inhibition was further confirmed to resensitize BCa cells to cytotoxic CD8 T cells. Conclusions: Modeling of the oxygen-deprived breast cancer TME in our engineered 3D physiologic and tumorous scaffolds supported known intratumoral hypoxia characteristics such as reduced BCa cell proliferation, increased ECM expression, and hindered immune infiltration. Furthermore, hypoxic inhibition was validated as a target to sensitize BCa cells to immune surveillance. These models could serve as a promising platform for the evaluation of immunologic events as well as a drug-screening platform tool to overcome hypoxia-driven immune evasion. Citation Format: Somshuvra Bhattacharya, Kristin Calar, Claire Evans, Pilar de la Puente. Modeling the oxygen-deprived breast cancer tumor microenvironment within a three-dimensional bioengineered platform that exhibits hypoxia-driven immune evasion [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B29.

BHLHE41 suppresses MCF-7 cell invasion via MAPK/JNK pathway.

Deregulation of the basic helix‐loop‐helix family member e41 (BHLHE41) has been characterized as a marker of progression of several cancers. In this study, we aimed to explore the mechanism by which BHLHE41 regulates the invasion of breast cancer cells. BHLHE41 suppresses, whereas the silencing of BHLHE41 promotes tumour invasion of both MCF‐7 and MDA‐MB‐231 cells. Meanwhile, BHLHE41 down‐regulated the transcription and translation of SNAI1, SNAI2, VIM and CDH2, and up‐regulated those of CLDN1, CLDN4 and CDH1. Reporter assay indicated that silencing of BHLHE41 dramatically activated the MAPK/JNK signalling pathway in MCF‐7 cell line and the hypoxia signalling pathway in MDA‐MB‐231 cell line. Furthermore, silencing of BHLHE41 activated the MAPK/JNK signalling pathway by up‐regulating phosphorylated JNK and failed to affect the expression of HIF‐1 alpha in MCF‐7 cells. After blocking the MAPK/JNK signalling pathway by specific inhibitor SP600125, silencing of BHLHE41 failed to promote tumour cell invasion. These results suggest that BHLHE41 facilitates MCF‐7 cell invasion mainly via the activation of MAPK/JNK signalling pathway. In conclusion, although BHLHE41 suppresses tumour invasion in MCF‐7 and MDA‐MB‐231 cell lines, the specific regulatory mechanisms may be different.

Hyperbaric Oxygen (HBO) Reduce Expression of Hypoxia Inducible Factor-1 Alpha (HIF-1 alpha) and Endometriotic Tissue Size in Mice Model of Endometriosis

Hypoxia in endometriosis will increase the expression of Hypoxia Inducible Factor-1alpha (HIF1alpha) and its expression could be decreased by Hyperbaric Oxygen (HBO). This study aimed to analyze the effect of HBO 2.4 ATA for 3x30 minutes per day for 10 days on the expression of HIF-1 alpha and endometriotic tissue size on mice model of endometriosis. This study was an experimental laboratory study with a separate pretest-posttest control group design. The mice were divided into three groups, the first was a pretest control group (which describes the condition after endometrium transplantation), the second was the endometriotic group that received hyperbaric oxygen, and the third was the endometriotic group that did not receive hyperbaric oxygen therapy. The endometriosis implant size in the peritoneal tissue was assessed and the immunohistochemistry examination was conducted to determine the expression of HIF-1 alpha. The endometriosis tissue size was reduced in the HBO group compared to the control and nonHBO group. The lowest expression of HIF-1 alpha was significantly found in HBO over the other group. The decrease of HIF1 alpha expression mediates the reduction of size endometriotic tissue due to the therapy of HBO.

Dexmedetomidine Regulates Proliferation, Apoptosis, Migration, and Invasion in Ovarian Cancer Cells via MiR-155-HIF-1α Axis.

BackgroundDexmedetomidine (DMED) is widely used as an adjuvant anesthetic, but how DMED regulates biological behavior of OC cells remains an area of active research. This study investigated the mechanism by which DMED regulates the proliferation, apoptosis, migration, and invasion abilities of OC cells.Material/MethodsWe determined the optimal concentration of DMED for use in treating SKOV3 cells. The biological activities of DMED-treated SKOV3 cells following transfection with miR-155 inhibitor or si-HIF-1α were measured by CCK-8 assay, flow cytometry, wound healing assay, and Transwell assay. qRT-PCR and Western blot analysis were performed to assess the expression levels of apoptotic-related caspase-3 and Mcl-1. Luciferase reporter assay verified the targeting relationship of miR-155 and HIF-1α.ResultmiR-155 was downregulated while HIF-1α was upregulated in SKOV3 cells. DMED dose-dependently reduced HIF-1α expression in SKOV3 cells, and upregulated the expression of miR-155. DMED inhibited the proliferation, migration and invasion abilities of OC cells, but also contributed to apoptosis of SKOV3 cells, while transfection of miR-155 inhibitor inhibited the effect of DMED on SKOV3 cells. In contrast, transfection with si-HIF-1α enhanced the effects of DMED on SKOV3 cells. HIF-1α was found to be a target gene of miR-155.ConclusionsOur results suggest that DMED blocks cell proliferation, migration, and invasion and accelerates cell apoptosis in OC.

Abstract C34: Signaling regulation of epithelial-mesenchymal transition in pancreas cancer cells cultured under hypoxic conditions

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit hypoxia due to high oxygen demand and oxygen diffusional limitations (1). Hypoxia has been reported as a driver of epithelial-mesenchymal transition (EMT) (2), which occurs early in pancreas cancer dissemination and promotes resistance to chemotherapy (3,4). Druggable signaling pathways that promote EMT in the hypoxic PDAC microenvironment may therefore represent useful targets for combination therapy. Our lab previously studied the roles of ERK and SHP2 in driving EMT in response to growth factors (5,6). More recently, our lab employed an unbiased data-driven modeling approach and found that the ERK, JNK, and NF-kappaB pathways coordinate to drive robust EMT in PDAC cells treated with combinations of growth factors (unpublished). In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to a shift from normoxic to hypoxic conditions. The immortalized human PDAC cell line HPAF-II, which exhibits baseline epithelial characteristics, was used for these studies. In simple two-dimensional cell culture, a shift from 20% to 1% oxygen for ≤120 hr led to several indications of EMT, including decreased epithelial cell cluster circularity, reduced junctional E-cadherin expression and an increased fraction of vimentin-positive cells (determined by an automated image analysis pipeline), and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2 (determined by RT-qPCR). A hypoxic cellular response was confirmed by immunoblotting, which showed an early (4 hr) peak in HIF-1alpha expression and a relatively late (48 hr) peak in HIF-2alpha expression, as well as increased expression of the HIF target genes PGK1 and SLC2A1. In three-dimensional cultures of HPAF-II cell spheroids in a collagen I matrix, a shift to hypoxia promoted cell invasion into the matrix, a behavior also observed when HPAF-II cells were treated with exogenous growth factors that drive EMT. To probe for the activation of the ERK, JNK, and NF-kappaB pathways, we used a combination of immunoblotting and immunofluorescence microscopy. A shift from 20% to 1% oxygen increased the phosphorylation of ERK and promoted the nuclear localization of c-Jun, a substrate of JNK. Inhibition of MEK or JNK at the same time as the shift to 1% oxygen prevented the loss of junctional E-cadherin that otherwise occurred, with JNK inhibition having the greater effect. JNK inhibition also abrogated loss of CDH1 transcripts and the increase of VIM transcripts otherwise observed in response to hypoxia. Thus, activation of the ERK and JNK pathways in PDAC cells in response to a shift to low oxygen conditions promoted EMT. These pathways may therefore represent useful targets for antagonizing EMT in the hypoxic microenvironment characteristic of PDAC tumors.

Effects of dietary yeast hydrolysate on the growth, antioxidant response, immune response and disease resistance of largemouth bass (Micropterus salmoides)

A 56-day growth trial was conducted to investigate the effects of dietary yeast hydrolysate on the growth performance, antioxidation, immune response and resistance against Aeromonas hydrophila in largemouth bass. Four experimental diets were prepared with yeast hydrolysate levels of 0% (Y0), 1.5% (Y1.5), 3.0% (Y3.0) and 4.5% (Y4.5). Each diet was randomly assigned to triplicate 150-L tanks and each tank was stocked with 30 largemouth bass (initial body weight, IBW = 7.71 ± 0.02 g). A challenge test was carried out after the feeding trial by injecting A. hydrophila intraperitoneally for 4-day observation. The results showed that the FBW and WGR in Y1.5 group were significantly higher than those in Y0 group (P < 0.05) and the feed conversion ratio (FCR) got the lowest value in Y1.5 group. And the hydrolysate supplement significantly increased the 4-day cumulative survival rate after the bacterial challenge (P < 0.05). The plasma malondialdehyde was lower in the yeast hydrolysate supplement groups in both pre- and post-challenge test (P < 0.05), while the plasma C3 increased (P < 0.05). In post-challenge test, the plasma superoxide dismutase (SOD) and catalase (CAT) activities increased in the Y1.5 and Y3.0 groups respectively (P < 0.05), and plasma lysozyme in Y1.5 group and the plasma IgM in Y3.0 group were higher than those in others respectively (P < 0.05). For the q-PCR results, in post-challenge test, the hepatic hep2 expression level in Y1.5 and Y4.5 groups were both significantly higher than those in others (P < 0.05), as well as il-8 in Y3.0 group. The spleen hif-1alpha and tgf-beta1 expression levels in Y4.5 group were all significantly lower than those in others (P < 0.05), while the gilt was significantly higher (P < 0.05) in the post-challenge test. And the expression levels of spleen tnf-alpah1 in Y1.5 and Y3.0 groups and il-8 in Y3.0 group were all significantly higher than those in other groups (P < 0.05) in the post-challenge test. The head kidney gilt expression level was significantly higher in the yeast hydrolysate supplement groups compared with the Y0 group (P < 0.05), and the head kidney il-8 expression level in Y1.5 group was significant higher than those in other groups in post-challenge test (P < 0.05). The present results indicated dietary yeast hydrolysate improved the antioxidant ability and enhanced the immune response of largemouth bass without negative effect on growth. And 1.5% or 3.0% of dietary yeast hydrolysate was recommended for largemouth bass based on the present results.

Angiopoietin-Like Protein 4 (ANGPTL4) Induces Retinal Pigment Epithelial Barrier Breakdown by Activating Signal Transducer and Activator of Transcription 3 (STAT3): Evidence from ARPE-19 Cells Under Hypoxic Condition and Diabetic Rats.

BackgroundDiabetic retinopathy is a primary contributor of visual impairment in adult diabetes mellitus patients. Diabetic retinopathy causes breakdown of blood retinal barrier (BRB), and leads to diabetic macular edema. Previous studies have demonstrated angiopoietin-like protein 4 (ANGPTL4) as an effective diabetic retinopathy therapeutic target, however, its role in maintaining the outer BRB in diabetic retinopathy has yet not elucidated.Material/MethodsWe established an in vivo diabetic rat model with the use of streptozotocin injections and cultured ARPE-19 cells under (hypoxia, 1%) condition. We first investigated the expression of hypoxia induced factor-1α (HIF-1α) and ANGPTL4 in vivo and subsequently studied the transcriptional regulation and underlying molecular mechanisms in ARPE-19 cells under oxygen-deprived situations.ResultsThe expression of HIF-1α and ANGPTL4 was increased with diabetic retinopathy progression both in vivo and in vitro. Depletion of HIF-1α by siRNA inhibited hypoxia-induced ANGPTL4 expression. Repressing the HIF-1α/ANGPTL4 signaling effectively alleviated the migration and cellular permeability induced by hypoxia in ARPE-19 cells. Depletion of ANGPTL4 by siRNA significantly alleviated signal transducer and activator of transcription 3 (STAT3) activity in vitro, thereby attenuating the decrease of tight junction proteins occludin and zona occludens-1 (ZO-1) under hypoxia in ARPE-19 cells.ConclusionsOur results suggest that ANGPTL4 partially modulates STAT3 and could serve as an effective diabetic retinopathy treatment strategy.

KDELR2 Promotes Glioblastoma Tumorigenesis Targeted by HIF1a via mTOR Signaling Pathway.

The KDEL (Lys-Asp-Glu-Leu) receptors (KDELRs), proteins with seven transmembrane domains, are primarily responsible for endoplasmic reticulum (ER) homeostasis. Recent studies have found additional function of KDELRs in growth, cellular secretory traffic, immune response, and autophagy; however, its role in tumorigenesis is still poorly understood. Here, we showed that KDELR2 is highly expressed in glioblastoma (GBM) tissues. Reviewing the expression of KDELR2 in TCGA and REMBRANDT database, we found that higher expression of KDELR2 is associated with shorter survival of GBM patients. We explored the effect of KDELR2 on tumorigenesis in GBM cells and animal model (nude mice), and identified KDELR2 as oncogene promoting cell proliferation. Additionally, KDELR2 expression in GBM cells correlated positively with HIF1alpha (HIF1α) expression, and we demonstrated by ChIP-qPCR and luciferase reporter assay that the upstream region of the KDELR2 gene is directly targeted by HIF1alpha. Taken together, our data suggest that KDELR2 is a target gene downstream of HIF1-alpha driving the malignancy of GBM and could eventually serve as a therapeutic target for the treatment of GBM patients.

Abstract 5154: Developing Micro-fluidic Chip to understand Altruistic stem cell reprogramming

Abstract Background:Tumor microenvironment (TME) is a complex network of cancer and stromal cell interactions in specialized niches. Cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) co-exist as significant components of TME that contribute to tumor progression. However, it is not clear whether MSCs compete or co-operate with CSCs for niche occupancy. In principle, stem cell competition eliminates the competing neighbors (e.g. p53 mutant cancer cells) while stem cell altruism enhances group-fitness in the niche. We found that the conditioned media of oral cancer CSCs, ABCG2+ cells, induced stem cell altruism in CD271+ MSCs (1). This indirect demonstration suggests the potential role of stem cell altruism in TME mediated tumor progression. Here we hypothesize that CSC mediated stem cell altruism of MSCs can be studied using an organ-on-chip microfluidic device. Methods:Naïve CD271+ MSCs were maintained in vitro by using an established culture method that we previously reported (2). Development of organ-on-chip microfluidic platform involved coating of 96 wells with polydimethylsiloxane (PDMS) and squalene to enhance cellular adhesion and delay stem cell differentiation. Two wells were linked by microfluidic channels to facilitate cell-to-cell interaction. We used 1000 ABCG2+ CSCs obtained from SCC-25 cell line (3) and equal numbers of naïve CD271+ MSCs. CSCs co-cultured with MSC in a Boyden-chamber assay was used as control population. After two weeks of culture, MSCs were evaluated for potential reprogramming to altruistic stem cell (ASC) phenotype as previously described. Results:We observed that MSCs collected from organ-on-chip exhibit stem cell altruism as indicated by loss of p53 and gain of HIF-2alpha expression as measured by In-Cell western assay (1). These MSCs also exhibited secretion of GSH, another characteristic of ASCs. Subsequently, these cells activated p53/MDM2 oscillation, and underwent apoptosis/differentiation, another important characteristic of ASCs. While, MSCs cultured in Boyden chamber assay, showed rapid proliferation and loss of CD271+ expression, and gain of markers associated with carcinoma-associated-fibroblasts including SMA and cytokeratin 8. Conclusion:Overall, our findings suggest that microfluidics-based organ-on-chip device can be used to understand stem cell altruism in TME. 1. PMID:22689594.2. PMID: 288841133. Talukdar J et al 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 920. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Sorra Sandhya, Joyeeta Talukdar, Lekhika Pathak, Hong Li, Bikul Das. Developing Micro-fluidic Chip to understand Altruistic stem cell reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5154.

CT Texture Analysis-Correlations With Histopathology Parameters in Head and Neck Squamous Cell Carcinomas.

Introduction: Texture analysis is an emergent imaging technique to quantify heterogeneity in radiological images. It is still unclear whether this technique is capable to reflect tumor microstructure. The present study sought to correlate histopathology parameters with texture features derived from contrast-enhanced CT images in head and neck squamous cell carcinomas (HNSCC).Materials and Methods: Twenty-eight patients with histopathological proven HNSCC were retrospectively analyzed. In every case EGFR, VEGF, Hif1-alpha, Ki67, p53 expression derived from immunhistochemical specimen were semiautomatically calculated. Furthermore, mean cell count was estimated. Texture analysis was performed on contrast-enhanced CT images as a whole lesion measurement. Spearman's correlation analysis was performed, adjusted with Benjamini-Hochberg correction for multiple tests.Results: Several texture features correlated with histopathological parameters. After correction only CT texture joint entropy and CT entropy correlation with Hif1-alpha expression remained statistically significant (ρ = −0.60 and ρ = −0.50, respectively).Conclusions: CT texture joint entropy and CT entropy were associated with Hif1-alpha expression in HNSCC and might be able to reflect hypoxic areas in this entity.

An Immunohistochemical Study of HIF-1 Alpha in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma.

To evaluate and compare the expression of HIF-1 Alpha (HIF-1α) in oral epithelial dysplasia (OED) and various grades of Oral squamous cell carcinoma (OSCC). 30 cases each of OEDand OSCC were stained with HIF-1α antibody. Quantification of HIF-1α positive cellswas carried out and the data was statistically analysed. The mean % HIF-1α labeling index (HIF-1α LI) increased significantly from mild OED (32.11%), moderate OED (55.07%), to severe OED (64.58%). There was a statistically significant increase in the expression of HIF-1α as grades of OED increased. The mean HIF-1α LI % in well differentiated OSCC was 46.3%, Moderately differentiated OSCC-76.31% and Poorly differentiated OSCC-89.9%. The mean HIF-1α LI was found to increase with increasing grades of OSCC which was statistically significant (P < 0.05). Further a comparison of mean HIF-1α LI in OED with different histologic grades of OSCC by Independent samples t test was performed. We found statistically significant difference between OED and moderately differentiated OSCC and OED and poorly differentiated OSCC (P = 0.000). Progressive increase in expression of HIF-1α was noted from OED to OSCC. It can be postulated that epithelial dysplastic lesions with increased HIF-1α expression are at greater risk of malignant transformation, suggesting that the expression of HIF-1α is an early event in oral carcinogenesis.

Associations between Histogram Analysis Parameters Derived from DCE-MRI and Histopathological Features including Expression of EGFR, p16, VEGF, Hif1-alpha, and p53 in HNSCC.

Background Our purpose was to elucidate possible correlations between histogram parameters derived from dynamic contrast-enhanced MRI (DCE-MRI) with several histopathological features in head and neck squamous cell carcinomas (HNSCC). Methods Thirty patients with primary HNSCC were prospectively acquired. Histogram analysis was derived from the DCE-MRI parameters: Ktrans, Kep, and Ve. Additionally, in all cases, expression of human papilloma virus (p16) hypoxia-inducible factor-1-alpha (Hif1-alpha), vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and tumor suppressor protein p53 were estimated. Results K ep kurtosis was significantly higher in p16 tumors, and Ve min was significantly lower in p16 tumors compared to the p16 negative tumors. In the overall sample, Kep entropy correlated well with EGFR expression (p=0.38, P=0.04). In p16 positive carcinomas, Ktrans max correlated with VEGF expression (p=0.46, P=0.04), Ktrans kurtosis correlated with Hif1-alpha expression (p=0.46, P=0.04), and Ktrans entropy correlated with EGFR expression (p=0.50, P=0.03). Regarding Kep parameters, mode correlated with VEGF expression (p=0.51, P=0.02), and entropy correlated with Hif1-alpha expression (p=0.47, P=0.04). In p16 negative carcinomas, Kep mode correlated with Her2 expression (p=−0.72, P=0.03), Ve max correlated with p53 expression (p=−0.80, P=0.009), and Ve p10 correlated with EGFR expression (p=0.68, P=0.04). Conclusion DCE-MRI can reflect several histopathological features in HNSCC. Associations between DCE-MRI and histopathology in HNSCC depend on p16 status. Kep kurtosis and Ve min can differentiate p16 positive and p16 negative carcinomas.