UNASSIGNED: Oral submucous fibrosis (OSMF) is a potentially malignant disorder associated with habit of chewing betel quid containing arecanut. Morphological features of OSMF especially fibrosis suggests a possibility of the hypoxic environment in diseased tissues. The adaptation of cells to hypoxia appears to be mediated via hypoxia inducible factor-1α (HIF-1α) which is also said to be associated with malignant transformation of epithelial cells in various other carcinomas like prostate and cervical carcinoma. Therefore, the main objective of this study was to investigate the role of HIF-1α in progression and malignant transformation of OSMF.
UNASSIGNED: The study group consisted of histo-pathologically diagnosed 30 cases of oral submucous fibrosis and 10 cases of OSCC were taken as control. The immunohistochemistry was carried out on neutral buffered formalin-fixed paraffin-embedded tissue sections by using the monoclonal antibody of HIF-1α. Statistical analysis was done using SPSS software version 2.0.
UNASSIGNED: A gradual and significant rise in the expression of HIF-1α was observed in various grades of OSMF and OSCC cases. HIF 1α expression was increased in cases showing hylanization and constricted blood vessels. A cut off value of 39.6% of HIF-1α positive cells was determined statistically to categorize the cases into high risk and low risk group for malignant transformation.
UNASSIGNED: Overexpression of HIF-1α may contribute to the progression and malignant transformation of OSMF. Cases expressing more than 40% of HIF-1α positive cells are at a greater risk for malignant transformation.

The hydrolytic stability, hemocompatibility, antioxidant properties and in vitro cytotoxic activity of {5-[(4,6-di(aziridin-1-yl)-1,3,5-triazin-2-yl)amino]-2,2-dimethyl-1,3-dioxan-5-yl}methyl 2-(5-phenyl-2H-tetrazol-2-yl)acetate have been studied. 1H NMR spectroscopy showed that this tetrazole-containing derivative of 1,3,5-triazine is stable in neutral (pH 7) and alkaline (pH 10) media; hydrolysis of the dioxane cycle occurs in an acidic environment (pH 3). It has been established that {5-[(4,6-di(aziridin-1-yl)-1,3,5-triazin-2-yl)amino]-2,2-dimethyl-1,3-dioxan-5-yl}methyl-2-(5-phenyl-2H-tetrazol-2-yl)acetate is hemocompatible, exhibits antioxidant properties, but does not show antiradical activity over the entire range of concentrations. In turn, the study of cytotoxic activity in vitro showed that the tetrazole-containing derivative of 1,3,5-triazine has an effect on the cell lines of human alveolar basal epithelium adenocarcinoma A549 (IC50 41.3 μmol l-1), human ovarian teratocarcinoma PA-1 (IC50 10.6 μmol l-1), hepatocarcinoma Huh7 (IC50 19.9 μmol l-1), cervical cancer HeLa (IC50 3.7 μmol l-1), and human embryonic kidney HEK293 (IC50 15.8 μmol l-1). It was suggested one of the possible mechanism of substance 2 cytotoxicity via HIF pathway inhibition.

Hypoxia and inflammation are intensely connected in a functional crosstalk. Within this crosstalk, two major transcription factors take center stage: HIF and NF-κB. To investigate transcription factor function, an important aspect is its ability to bind DNA. The most appropriate method to study this property in cells is the use of chromatin immunoprecipitation followed by qPCR and/or next generation sequencing. This allows identification of potentially directly regulated genes as well as enhancer regions. Here we describe the ChIP-qPCR method in detail, including key aspects important for the success of the technique.

(1) Background: Triple negative breast cancer (TNBC) is a highly aggressive tumor, associated with high rates of early distant recurrence and short survival times, and treatment may require surgery, and thus anesthesia. The effects of anesthetic drugs on cancer progression are under scrutiny, but published data are controversial, and the involved mechanisms unclear. Anesthetic agents have been shown to modulate several molecular cascades, including PI3K/AKT/mTOR. AKT isoforms are frequently amplified in various malignant tumors and associated with malignant cell survival, proliferation and invasion. Their activation is often observed in human cancers and is associated with decreased survival rate. Certain anesthetics are known to affect hypoxia cell signaling mechanisms by upregulating hypoxia-inducible factors (HIFs). (2) Methods: MCF-10A and MDA-MB 231 cells were cultivated and CellTiter-Blue® Cell Viability assay, 2D and 3D matrigel assay, immunofluorescence assays and gene expressions assay were performed after exposure to different sevoflurane concentrations. (3) Results: Sevoflurane exposure of TNBC cells results in morphological and behavioral changes. Sevoflurane differently influences the AKT isoforms expression in a time-dependent manner, with an important early AKT3 upregulation. The most significant effects occur at 72 h after 2 mM sevoflurane treatment and consist in increased viability, proliferation and aggressiveness and increased vimentin and HIF expression. (4) Conclusions: Sevoflurane exposure during surgery may contribute to cancer recurrence via AKT3 induced epithelial-mesenchymal transition (EMT) and by all three AKT isoforms enhanced cancer cell survival and proliferation.

Daprodustat is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis and regulates genes related to iron metabolism. The efficacy (noninferiority) and safety of daprodustat compared with standard therapy (darbepoetin alfa) was evaluated.
This was a randomized, phase 3, double-blind, active-control study in Japanese patients receiving hemodialysis with anemia of CKD. Participants\' treatment was switched from current erythropoiesis-stimulating agents (ESAs) to daprodustat 4 mg once daily or darbepoetin alfa 10-60 μg once weekly (on the basis of the prestudy ESA dose). Dose was adjusted every 4 weeks for daprodustat or every 2 weeks for darbepoetin alfa, according to a protocol-specified algorithm. The primary end point was mean hemoglobin during weeks 40-52 in the intent-to-treat population.
Of 332 participants screened, 271 participants were randomized (safety evaluation: 271 participants; efficacy evaluation: 267 intent-to-treat population). The mean hemoglobin during weeks 40-52 were maintained within the target range in both groups (10.9 g/dl [95% confidence interval (95% CI), 10.8 to 11.0] for daprodustat, and 10.8 g/dl [95% CI, 10.7 to 11.0] for darbepoetin alfa). Daprodustat was noninferior to darbepoetin alfa, as the lower bound of the confidence interval for the treatment difference (0.1 g/dl; 95% CI, -0.1 to 0.2 g/dl) was greater than the noninferiority criterion of -1.0 g/dl. For most participants, hemoglobin was maintained within the target range (10.0-12.0 g/dl) during weeks 40-52 (88% daprodustat; 90% darbepoetin alfa). Geometric mean hepcidin levels decreased more at week 52 with daprodustat (-37%; 95% CI, -49 to -23) than with darbepoetin alfa (-20%; 95% CI, -36 to -1), and an increase in total iron-binding capacity was observed in the daprodustat group. Frequency of adverse events were generally similar between daprodustat and darbepoetin alfa.
Oral daprodustat was noninferior to darbepoetin alfa as measured by mean hemoglobin over weeks 40-52 in Japanese patients receiving hemodialysis switched from ESAs.
201754, Clinicaltrials.gov, NCT02969655.

BACKGROUND: The present study was designed to investigate the effects of Berberis vulgaris (BV) juice consumption on plasma levels of insulin-like growth factor (IGF-1), IGF-binding proteins (IGFBPs), and the expression of PPAR-γ, VEGF and HIF in women with benign breast disease.
METHODS: This parallel design randomized, double-blind controlled clinical trial was conducted on 85 eligible patients diagnosed with benign breast disease. They were assigned randomly into either BV juice group (n = 44, BV juice: 480 ml/day) or placebo group (n = 41, BV placebo juice: 480 ml/day) for 8 weeks intervention. Participants, caregivers and those who assessed laboratory analyses were blinded to the assignments. Plasma levels of biomarkers were measured at baseline and after 8 weeks by ELISA. Quantitative real-time PCR was used to measure the fold change in the expression of each interested gene.
RESULTS: The compliance of participants was 95.2% and 40 available subjects analyzed in each group at last. Relative treatment (RT) effects for BV juice caused 16% fall in IGF-1 concentration and 37% reduction in the ratio of IGF-1/1GFBP1. Absolute treatment effect expressed 111 ng/ml increased mean differences of IGFBP-3 between BV group and placebo. Plasma level of PPAR-γ increased in both groups but it was not significant. Fold changes in the expressions of PPAR-γ, VEGF and HIF showed down-regulation in the intervention group compared to placebos (P < 0.05).
CONCLUSIONS: The BV juice intervention over 8 weeks was accompanied by acceptable efficacy and decreased plasma IGF-1, and IGF-1/IGFBP-1 ratio partly could be assigned to enhanced IGFBP-1 level in women with BBD. The intervention caused reductions in the expression levels of PPAR, VEGF, and HIF which are remarkable genomic changes to potentially prevent breast tumorigenesis.
BACKGROUND: IRCT2012110511335N2. Registered 10 July 2013 (retrospectively registered).

The house gecko (Hemidactylus platyurus) has evolved the ability to autotomize its tail when threatened. The lost part is then regrown via epimorphic regeneration in a process that requires high energy and oxygen levels. Oxygen demand is therefore likely to outstrip supply and this can result in relative hypoxia in the tissues of the regenerating tail. The hypoxic state is stabilized by the Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α proteins. We induced tail autotomy in 30 mal H. platyurus adults using a standard procedure and then collected samples of the regenerated tail tissue on days 1, 3, 5, 8, 10, 13, 17, 21, 25, and 30 post autotomy. For each sample, mRNA expression was analyzed by qPCR, proteins were analyzed using Western Blot tests and immunohistochemistry, and the histological structure was analyzed using Hematoxylin and Eosin staining. On day 1, HIF-1α mRNA expression increased and the tissue was dominated by leucocyte and erythrocyte cells. HIF-1α mRNA expression peaked on day 3, at which time some cells were actively proliferating, migrating, and differentiating. At the same time as HIF-1α expression decreased, HIF-2α mRNA expression increased, as did overall cellular activity. HIF-2α expression increased more gradually but was present over a longer period of time than HIF-1α. We hypothesize that HIF-1α helps to initially stimulate the tissue regeneration process while HIF-2α functionally takes over the role of HIF-1α after HIF-1α succumbs to the oxygen conditions, but we suspect that both HIF-1α and HIF-2α play a role in overcoming the tissue\'s hypoxic state.

UNASSIGNED: Hypoxia is a major factor in prostate cancer aggressiveness and radioresistance. Predicting which patients might be bad candidates for radiotherapy may help better personalize treatment decisions in intermediate-risk prostate cancer patients. We assessed spatial distribution of 18F-Misonidazole (FMISO) PET/CT uptake in the prostate prior to radiotherapy treatment.
UNASSIGNED: Intermediate-risk prostate cancer patients about to receive high-dose (>74 Gy) radiotherapy to the prostate without hormonal treatment were prospectively recruited between 9/2012 and 10/2014. Prior to radiotherapy, all patients underwent a FMISO PET/CT as well as a MRI and 18F-choline-PET. 18F-choline and FMISO-positive volumes were semi-automatically determined using the fuzzy locally adaptive Bayesian (FLAB) method. In FMISO-positive patients, a dynamic analysis of early tumor uptake was performed. Group differences were assessed using the Wilcoxon signed rank test. Parameters were correlated using Spearman rank correlation.
UNASSIGNED: Of 27 patients (median age 76) recruited to the study, 7 and 9 patients were considered positive at 2.5h and 3.5h FMISO PET/CT respectively. Median SUVmax and SUVmax tumor to muscle (T/M) ratio were respectively 3.4 and 3.6 at 2.5h, and 3.2 and 4.4 at 3.5h. The median FMISO-positive volume was 1.1 ml.
UNASSIGNED: This is the first study regarding hypoxia imaging using FMISO in prostate cancer showing that a small FMISO-positive volume was detected in one third of intermediate-risk prostate cancer patients.

Exposing cells to a hypoxic environment leads to significant physiological and molecular alterations. Most of the hypoxic responses are regulated by the transcription factors known as hypoxia-inducible factors (HIFs). HIF1, a heterodimer of hypoxia-stabilized subunit HIF-1alpha and a constitutively expressed subunit HIF-1beta, serves as a key transcription factor that regulates gene expressions which are involved in cell growth, metabolism, and proliferation. The global expression patterns can be analyzed by utilizing RNA-Seq to understand the cellular alterations in hypoxia. This technique enables us to understand the comprehensive regulation of gene expression by specific factors or environmental stimuli. Here, we describe the complete process of studying hypoxia-mediated gene expression by using RNA-Seq, including the hypoxic treatment of cells, RNA isolation, RNA quality check, cDNA library preparation, and library quality check.

Hypoxia-inducible-factor-1α (HIF-1α) and HIF-1 degrading prolyl-hydroxylases (PHD) are key regulators of the hypoxic-inflammatory response. Functionally active genetic variants in the HIF-1α (C/T; Single Nucleotide Polymorphism (SNP) rs11549465) and the PHD2 gene (EGLN1; C/T; SNP rs516651 and T/C; SNP rs480902) are associated with altered HIF-1α mRNA nuclear translocation and an altered adaptation to hypoxia. Furthermore, the HIF system is important in surviving inflammatory disorders and sepsis. Thus, we tested the hypotheses, that SNPs in the HIF-1α or PHD2 genes are (1) common in Caucasians, with 2) the HIF-1α genetic variant being associated with an altered HIF-1α mRNA expression; and 3) independent risk factors for 30-day mortality in severe sepsis.
After ethics approval, 128 septic patients (Caucasian descent) were included prospectively within 24 h after first diagnosing sepsis. Patients characteristics and severity of illness (simplified acute physiology score II), genotypes (Taqman assay), and their influence on leukocyte HIF-1α-mRNA-expression (Real-Time PCR) and 30-day mortality were determined.
Frequencies were 0.8 % for homozygous HIF-1α TT-carriers (CT 17.6 %; CC 81.6 %), 2.5 % for homozygous PHD2 SNP rs516651 TT-allele carriers (CT 17.5 % and CC 80 %), and 9.4 % for homozygous PHD2 SNP rs480902 TT-allele carriers (CT 34.4 % and CC 56.3 %). While HIF-1α T-allele carriers had a borderline decrease in HIF-1α-mRNA-expression (p = 0.06) neither HIF-1α nor PHD2 SNPs were (independent) risk factors for 30-day mortality.
Genetic variants in HIF-1α and PHD2 genes exist in Caucasians but do not appear to alter 30-day mortality in sepsis.