Animals ; Humans ; Hypoxia ; metabolism ; Hypoxia-Inducible Factor 1 ; genetics ; metabolism ; Neovascularization, Pathologic ; metabolism

To study the expression of hypoxia inducible factor-1alpha (HIF-1alpha) protein in prostate cancer (Pca) and its biological significance, the expression of HIF-1alpha was assayed by means of immunohistochemical technique in 42 prostate cancer, 12 prostatic intraepithelial neoplasm (PIN) and 9 normal prostate tissue (NP) specimens. Western blot was used to examine the expression of HIF-1alpha in prostate cancer cell line (PC-3M) induced by different oxygen tension. HIF-1alpha expression was positive in 33 Pca and 9 PIN specimens, and the positive rate of HIF-1alpha was higher in distant metastasis patients than in patients without metastasis of prostate cancer (P<0.05), while there was no expression of HIF-1alpha in NP. The level of HIF-1alpha in PC-3M significantly increased with the decrease of oxygen tension (P<0.01). Overexpression of HIF-1alpha is the preliminary event of the formation of Pca, which may induce carcinoma into malignant phenotype. Thus it may serve as an early diagnosis marker and the novel target for Pca treatment.
Adenocarcinoma/*metabolism ; Cell Line, Tumor ; Hypoxia-Inducible Factor 1, alpha Subunit/*biosynthesis ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; Prostatic Neoplasms/*metabolism ; Tumor Markers, Biological/*biosynthesis

OBJECTIVETo detect the expression of hypoxia-inducible factor-1alpha(HIF-1alpha) in endometriosis and explore the possible role of HIF-1alpha in the pathogenesis of endometriosis.

METHODSImmunohistochemistry was performed to examine the expression of HIF-1alpha in 20 normal endometrium, 20 ectopic endometrium and 68 eutopic endometrium specimens from 68 endometriosis patients, and the results were analyzed statistically.

RESULTSThe expression of HIF-1alpha was significantly increased in ectopic endometrium than in normal endometrium (P<0.01), and the expression did not undergo changes with the normal menstrual cycle in the three types of endometrium.

CONCLUSIONHIF-1alpha expression increases in ectopic endometrium, suggesting that HIF-1alpha plays an important role in the pathogenesis of endometriosis.

Adult ; Endometriosis ; genetics ; Endometrium ; metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Menstrual Cycle ; metabolism

OBJECTIVETo study the effects of oxygen and calcium on the expression of eukaryotic vectors harboring wild-type or mutated hypoxia-inducible factor-1alpha (HIF-11alpha) in HEK293 cells.

METHODSHEK293 cells were transiently transfected with pcDNA3.1+/HIF-11alpha, pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha-564Ala-803Ala via lipofectin. Western blotting were used to detect HIF-11alpha protein after normoxic or hypoxic exposure of the transfected HEK293 cells in the presence or absence of Ca(2+). The levels of vascular endothelial growth factor (VEGF) mRNA in the transfected cells in normoxic condition was detected using RT-PCR.

RESULTSThe levels of HIF-11alpha protein and VEGF mRNA increased in HEK293 cells transfected with the vectors harboring mutated HIF-11alpha, but not in the cells transfected with wild-type HIF-11alpha vectors in normoxia. Hypoxia increased the levels of HIF-11alpha protein in the cells transfected with wild-type HIF-11alpha vectors, which was inhibited by the application of Ca(2+). Ca(2+) showed no inhibitory effect on HIF-11alpha levels in HEK293 cells transfected with the vectors containing mutated HIF-11alpha.

CONCLUSIONThe protein products of pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha- 564Ala-803Ala in HEK293 cells enhance the cell tolerance to oxygen and protease.

Calcium ; metabolism ; Cell Hypoxia ; Genetic Vectors ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Oxygen ; metabolism ; RNA, Messenger ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

Gene Expression ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism

OBJECTIVETo investigate the effects of hypoxia induced by cobalt chloride on the expression and the activity of matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC-T6) and to clarify the possible mechanisms.

METHODSHSC-T6 cell line was grown in Dulbecco's modified Eagle medium with 10% fetal calf serum at 37 degrees C and 5% CO2. When reaching confluence, the cells were incubated with serum-free medium in the presence of cobalt chloride (0, 50, 100, 200 micromol/L) for six hours, and then the supernatant and the cells were harvested. The expression of the MMP-2 mRNA and HIF-1alpha protein in HSC-T6 cells was detected using RT-PCR and Western blot respectively. The activity of the MMP-2 in the supernatant was detected by zymography. The binding reaction between HIF-1a protein and MMP-2 gene sequence was investigated by electrophoresis mobility shift assay.

RESULTSWhen the concentration of CoCl2 increased from 0 micromol/L to 200 micromol/L, the expressions of MMP-2 mRNA (the rate of light density) were increased from 0.53+0.12 to 1.57+0.11 and the differences among these four groups were significant (F=34.21). The activity of MMP-2 (the value of light density*band area) decreased gradually from 84.49+5.38 to 53.70+3.42, and the differences among these four groups were also significant (F=29.54). The expressions of HIF-1a were increased gradually with the increase of the CoCl2 concentration. The shift band in the lane of the nuclear protein extraction and the MMP-2 probe containing hypoxia response element showed delays when compared with the lane of the sole probe, and the binding was partially abolished when competing sense oligonucleotides were used.

CONCLUSIONSOur results suggest that chemical hypoxia can up-regulate the expression of MMP-2 mRNA and decrease the activity of the enzyme. HIF-1alpha may play a part in the regulation of MMP-2 transcription under hypoxic conditions.

Animals ; Cell Hypoxia ; Cell Line ; Cobalt ; pharmacology ; Hepatic Stellate Cells ; enzymology ; Hypoxia ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; RNA, Messenger ; genetics ; Rats

BACKGROUNDEndometriosis (EMs) is a common gynecological disorder characterized by endometrial-like tissue outside the uterus. Hypoxia induces the expression of many important downstream genes to regulate the implantation, survival, and maintenance of ectopic endometriotic lesions. Transforming growth factor-beta 1 (TGF-β1) plays a major role in the etiology of EMs. We aimed to determine whether TGF-β1 affects EMs development and progression and its related mechanisms in hypoxic conditions.

METHODSEndometrial tissue was obtained from women with or without EMs undergoing surgery from October, 2015 to October, 2016. Endometrial cells were cultured and then exposed to hypoxia and TGF-β1 or TGF-β1 inhibitors. The messenger RNA (mRNA) and protein expression levels of TGF-β1, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF-1α) were measured. A Dual-Luciferase Reporter Assay was used to examine the effect of TGF-β1 and hypoxia on a VEGF promoter construct. Student's t-test was performed for comparison among groups (one-sided or two-sided) and a value of P < 0.05 was considered statistically significant.

RESULTSTGF-β1, VEGF, HIF-1α mRNA, and protein expression were significantly higher in EMs tissue than that in normal endometrial tissue (t = 2.16, P = 0.042). EMs primary cultured cells exposed to hypoxia expressed 43.8% higher VEGF mRNA and protein (t = 6.84, P = 0.023). VEGF mRNA levels increased 12.5% in response to TGF-β, whereas the combined treatment of hypoxia/TGF-β1 resulted in a much higher production (87.5% increases) of VEGF. The luciferase activity of the VEGF promoter construct was increased in the presence of either TGF-β1 (2.6-fold, t = 6.08, P = 0.032) or hypoxia (11.2-fold, t = 32.70, P < 0.001), whereas the simultaneous presence of both stimuli resulted in a significant cooperative effect (18.5-fold, t = 33.50, P < 0.001).

CONCLUSIONSThe data support the hypothesis that TGF-β1 is involved in the pathogenesis of EMs through regulating VEGF expression. An additive effect of TGF-β1 and hypoxia is taking place at the transcriptional level.

Blotting, Western ; Cells, Cultured ; Endometriosis ; genetics ; metabolism ; Female ; Humans ; Hypoxia ; genetics ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effects of hypoxia on sumoylation of HIF-1α, as well as transcriptional activity and protein stability of HIF-1α in Jurkat cells, and explore its effect and significance on modulating the transcriptional activity of down stream target gene.

METHODSCoCl(2) was used as a chemical inducer to simulate the hypoxia environment. Real-time fluorescence quantitative PCR and western blot were used to evaluate transcriptional activity and protein stability of HIF-1α, levels of SUMO-1 and SENP1 protein, and gene transcripts of VEGF, mdr1, mdr3, Mcl-1 and survivin respectively.

RESULTSAfter 4 h, 8 h, 16 h, 24 h and 72 h after hypoxia induction, the gene transcripts of HIF-1α were 0.79 ± 0.19, 2.65 ± 2.05, 4.19 ± 4.72, 2.77 ± 3.37, 0.09 ± 0.05 and 0.69 ± 0.55-fold (P > 0.01) of that of 0h in Jurkat cells, respectively, while the protein stability increased first, then decreased (P < 0.01). SENP-1 protein up-regulated first, then down-regulated, and the SUMO-1 protein changed in an opposite trend. Excepting for survivin gene, transcriptional activities of VEGF, mdr1, mdr3, and Mcl-1 were affected by the stability of HIF-1α protein.

CONCLUSIONHypoxia induces changes in SENP-1 expression, which increases the stability of HIF-1α by decreasing the sumoylation of HIF-1α and affects biological process by regulating the transcriptional activities of VEGF, mdr1, mdr3 and Mcl-1 gene.

Cell Hypoxia ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Jurkat Cells ; RNA, Messenger ; genetics ; Sumoylation ; Transcriptional Activation ; Vascular Endothelial Growth Factor A ; metabolism

AIMTo investigate the expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) in rats with chronic pulmonary hypertension induced by hypoxia and hypercapnia and its relationship with nitric oxide(NO).

METHODSFourty male Sprague-Dawley rats were randomly divided into four groups, normal control group (NC), hypoxia-hypercapnia group (HH), hypoxia - hypercapnia + L-arginine liposome group(HP) and hypoxia-hypercapnia+ N-nitro-L-arginine methylester group (HM). Colorimetric analysis, immunohistochemistry and in situ hybridization were used for detection of NO, HIF-1alpha and constitutive nitric oxide synthase (ecNOS).

RESULTS(1) The mean pulmonary arterial pressure (mPAP) and the weight ratio of right ventricular to left ventricle plus septum (RV/(LV + S)) of HH group were higher than those of NC group (P < 0.05), HP group much lower than HH group (P < 0.01), mPAP of HM higher than HH group ( P < 0.05). 2)0 Contents of NO in plasma and pulmonary tissue homogenates of HH group were much lower than those of NC group (P < 0.01), HP group higher than HH group (P < 0.01). There were no difference between HM group and HH group. (Expression of HIF-1alpha and HIF-1alpha mRNA in pulmonary arterioles of HH group were significantly higher than those of NC group( P < 0.01), HP group lower than HH group (P < 0.01) ,HM group higher than HH group (P < 0.01); Whereas expression of ecNOS and ecNOS mRNA in pulmonary arterioles of HH were lower than those of NC group( P < 0.05, IP group higher than HH group (P < 0.01), HM group lower than HH group (P < 0.05).

CONCLUSIONHIF-1alpha is involved in the pathogenesis of chronic pulmonary hypertension induced by hypoxia and hypercapnia. The protective function of NO in the pathogenesis might be partly depended on its effects on the expression/activity of HIF-1alpha in lung.

Animals ; Hypertension, Pulmonary ; etiology ; metabolism ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Anemia ; metabolism ; physiopathology ; Animals ; Cell Hypoxia ; Genetic Therapy ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; metabolism ; physiology ; Neovascularization, Pathologic ; metabolism ; pathology ; Neovascularization, Physiologic ; Osteogenesis ; RNA, Messenger ; metabolism