HIF-1alpha mRNA Levels Research Articles

Upregulation of Hypoxia-Inducible Factor-1α mRNA and its Clinical Significance in Non-small Cell Lung Cancer

Hypoxia-inducible factor 1alpha (HIF-1alpha) is a transcription factor that plays an important role in tumor growth by regulating the energy metabolism and angiogenesis. We herein investigated the mRNA expression level of HIF-1alpha in non-small cell lung cancer (NSCLC) tissues to clarify the impact on the clinical aspects of NSCLC patients.HIF-1alpha mRNA derived from either a tumor or an adjacent lung tissue was quantified using quantitative reverse transcription polymerase chain reaction in 66 patients with NSCLC. The relationship between the mRNA expression level of HIF-1alpha and clinicopathological factors was investigated.The expression level of HIF-1alpha mRNA, which correlated with its protein level, was significantly higher in tumor tissue than in the corresponding nontumor-bearing lung tissue (4.22 x 10(4) +/- 4.99 x 10(4) versus 1.24 x 10(4) +/- 1.15 x 10(4); p < 0.001). The level of HIF-1alpha mRNA showed a significantly positive correlation with the mRNA levels of vascular endothelial growth factor and type II hexokinase in tumors (p < 0.0001 for each). In node-negative patients, high expression levels of HIF-1alpha mRNA in tumors were associated with a poor prognosis (p = 0.0401), but not in the node-positive cases.The expression of HIF-1alpha mRNA is associated with disease progression in NSCLC tissues, and is expected as a biomarker or therapeutic target.

Increase in gene dosage is a mechanism of HIF‐1α constitutive expression in head and neck squamous cell carcinomas

The HIF-1alpha protein plays a key role in the cellular response to hypoxia via transcriptional regulation of genes involved in erythropoiesis, angiogenesis, and metabolism. Overexpression of HIF-1alpha is commonly found in solid tumors in significant association with increased patient mortality and resistance to therapy. The predominant mode of HIF-1alpha regulation by hypoxia occurs at the level of protein stability. In addition to hypoxia, HIF-1alpha protein stability and synthesis is regulated by nonhypoxic signals such as inactivation of tumor suppressors and activation of oncogenes. Here, we show that an increase in gene dosage may contribute to HIF-1alpha mRNA and protein overexpression in a nonhypoxic environment in head and neck squamous cell carcinomas (HNSCC). Increased HIF-1alpha gene dosage was found in one out of five HNSCC-derived cell lines and three out of 27 HNSCC primary tumors. Significantly, increased gene dosage in those samples was associated with high HIF-1alpha mRNA and protein levels. Normoxic overexpression of HIF-1alpha protein in HNSCC-derived cell lines was also paralleled by higher expression levels of HIF-1alpha target genes. Array CGH analysis confirmed the copy number increase of HIF-1alpha gene and revealed that the gene is contained within a region of amplification at 14q23-q24.2 both in the cell line and primary tumors. In addition, FISH analysis revealed the presence of 11-13 copies on a tetraploid background in SCC2 cells. These data suggest that increased HIF-1alpha gene dosage is a mechanism of HIF-1alpha protein overexpression in HNSCC that possibly prepares the cells for a higher activity in an intratumoral hypoxic environment.

Gefitinib circumvents hypoxia-induced drug resistance by the modulation of HIF-1α

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcriptional factor which is activated by hypoxia and associated with cell survival, proliferation and drug resistance. Recent studies have shown that the down-stream molecules of the epidermal growth factor receptor (EGFR) signal are involved in the hypoxia-dependent or -independent HIF-1alpha protein accumulation. Thus, we hypothesized that an EGFR-TK inhibitor, gefitinib, might circumvent the hypoxia-induced drug resistance via the regulation of HIF-1alpha expression. In our data, treatment of gefitinib suppressed induced HIF-1alpha by hypoxia. This action of gefitinib was caused by reduced protein stability without any change in the level of HIF-1alpha mRNA. The effect of gefitinib on down-regulation of HIF-1alpha was reversed by transfection of constitutively active form of Akt. The cellular response to gefitinib was similar in both normoxia and hypoxia condition. However, the response to conventional chemotherapeutic drugs decreased >50% under hypoxia condition and they did not change HIF-1alpha expression. In addition, the suppression of HIF-1alpha using siRNA overcame partially hypoxia-induced drug resistance. In conclusion, gefitinib was able to circumvent hypoxia-induced drug resistance suggesting that the effective suppression of HIF-1alpha by the inhibition of EGFR-Akt pathway may overcome the hypoxia-induced drug resistance.

Mechanism of regulation of leptin expression in human lung adenocarcinoma cells by hypoxia inducible factor-1alpha: a preliminary study

To investigate the regulation of leptin expression in human lung adenocarcinoma cells by hypoxia inducible factor-1 (HIF-1alpha) and the mechanism thereof. Lung adenocarcinoma tissues were collected from 42 patients during operation and samples of corresponding adjacent lung tissue were obtained from 16 of the 42 specimens. The expression levels of HIF-1alpha and leptin were detected by immunohistochemical staining. Human lung adenocarcinoma cells of the line A549 cells were cultured for 0, 12, 24, and 48 h respectively, exposed to hypoxia induced by CoCl2. Other A549 cells were treated with GL331, a kind of HIF-1alpha inhibitor, of the concentrations of 0, 5, 10, or 20 micromol/L under normoxic condition for 24 h, and then were exposed to hypoxia induced by CoCl2 for 24 h. RT-PCR and Western-blotting were used to examine the mRNA and protein expression levels of HIF-1alpha and leptin in different groups. Immunohistochemistry showed that the positive rates of HIF-1alpha and leptin in the lung adenocarcinoma tissue were 57.1% and 69.0% respectively, both significantly higher than those in the adjacent normal lung tissues (18.8% and 25.0% respectively, both P<0.01). The protein expression levels of HIF-1alpha in the hypoxia 0, 12, 24, and 48 h groups were 0.314+/-0.030, 0.552+/-0.027, 0.743+/-0.015, and 0.799+/-0.010 respectively, and the protein expression levels of leptin were 0.398+/-0.016, 0.633+/-0.036, 0.796+/-0.008, and 0.942+/-0.088 respectively, increasing in a time dependent manner too. The mRNA expression levels of leptin in the 4 hypoxia groups were 0.144+/-0.009, 0.336+/-0.017, 0.524+/-0.013, and 0.671+/-0.021 respectively, increasing in a time dependent manner, however, there was no significant differences in the mRNA expression levels of HIF-1alpha among the groups exposed to hypoxia for different courses of time (all P>0.05). The protein expression levels of HIF-1alpha and leptin, and the mRNA expression levels of leptin in the groups exposed to hypoxia for different courses of time were all significantly higher than those of the control (0 h hypoxia) group (all P<0.01). The mRNA and protein expression levels of leptin in the A549 cells exposed to GL331 and hypoxia were decreased dose-dependently. The expression of leptin is up-regulated by hypoxia and regulated by HIF-1alpha.

Apoptosis signal-regulating kinase 1 (ASK1) and HIF-1α protein are essential factors for nitric oxide-dependent accumulation of p53 in THP-1 human myeloid macrophages

Nitric oxide (NO) is a reactive secondary mediator, which has been found to participate in cell cycle regulation and apoptosis in myeloid macrophages, the key effectors of inflammatory and innate immune responses. However, the molecular mechanisms of nitric oxide-induced death of myeloid macrophages are not well understood. In this study we have found that NO derived from S-nitrosoglutathione (GSNO) activates ASK1 in THP-1 human myeloid macrophages in a concentration and time-dependent manner. It also induces accumulation of HIF-1alpha protein in a concentration-dependent manner, which peaks at 4 h of exposure to 1 mM GSNO. GSNO does not affect the level of HIF-1alpha mRNA as detected by the RT-PCR. In addition, GSNO was found to induce accumulation of p53 in normal but not HIF-1alpha knockdown THP-1 cells, where expression of this protein was silenced by specific siRNA. It has also been found that GSNO-mediated accumulation of p53 depends on activation of ASK1 since no GSNO-induced p53 stabilisation was observed in THP-1 cells transfected with dominant-negative form of this kinase. However, in both HIF-1alpha knockdown THP-1 cells and those transfected with the dominant-negative form of ASK1, GSNO was able to induce cell death as detected by the MTS cell viability assay leading to an increase in release of LDH.

Activation of HIF‐1α in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway

Accumulation of HIF-1alpha during normoxic conditions at high cell density has previously been shown to occur and can be used to stabilize HIF-1alpha protein in the absence of a specific anaerobic chamber. However, the impact and origin of this pool of HIF-1alpha, obtained under normoxia, has been underestimated. In this study, we have systematically compared the related pools of HIF-1alpha stabilized in normoxia by high cell density to those obtained at low density in hypoxia. At first glance, these two stimuli appear to have similar outcomes: HIF-1alpha stabilization and induction of HIF-1-dependent genes. However, upon careful analysis, we observed that molecular mechanisms involved are different. We clearly demonstrate that density-dependant HIF-1alpha accumulation during normoxia is due to the cells high consumption of oxygen, as demonstrated by using a respiration inhibitor (oligomycin) and respiratory-defective mutant cells (GSK3). Finally and most importantly, our data indicate that a decrease in AKT activity followed by a total decrease in p70(S6K) phosphorylation reflecting a decrease in mTOR activity occurs during high oxygen consumption, resulting from high cell density. In contrast, hypoxia, even at severe low O(2) levels, only slightly impacts upon the mTOR pathway under low cell density conditions. Thus, activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway whereas HIF-1alpha activation obtained in high confluency is totally dependent on mTOR pathway as rapamycin totally impaired (i) HIF-1alpha stabilization and (ii) mRNA levels of CA9 and BNIP3, two HIF-target genes.

Feiyanning Decoction down-regulates the expressions of phosphorylated Akt and hypoxia inducible factor-1α proteins in transplanted lung adenocarcinoma in nude mice

To observe the effects of Feiyanning Decoction, a compound traditional Chinese herbal medicine, in inhibiting the growth of transplanted A549 tumors in nude mice and the expressions of p-Akt and HIF-1alpha in A549 tumors. Thirty-two nude mice were transplanted with human lung adenocarcinoma cells (A549 cells) to make the mice model, and then randomly divided into four groups: untreated group (n=8), diamminedichloroplatinum (DDP) group (n=8), Feiyanning group (n=8) and DDP plus Feiyanning group (n=8). The nude mice in DDP group and DDP plus Feiyanning group were treated with DDP 0.1 mg by peritoneal injection on the 1st, 3rd and 5th day after model establishment. The nude mice in the Feiyanning group and the DDP plus Feiyanning group were treated with Feiyanning Decoction for 21 days. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of HIF-1alpha mRNA in tumors, and immunohistochemical method was used to detect the expressions of p-Akt and HIF-1alpha proteins in tumors. The inhibition rates of tumor in Feiyanning group and DDP plus Feiyanning group were 34.17% and 64.38% respectively. RT-PCR showed that the expression level of HIF-1alpha mRNA in DDP plus Feiyanning group was lower than that in the untreated group (P<0.05). Immunohistochemical staining showed that the optical density (OD) indexes of HIF-1alpha in DDP group, Feiyanning group and DDP plus Feiyanning group were lower than that in the untreated group (P<0.01). The expressions of p-Akt in the Feiyanning group and DDP plus Feiyanning group were lower than that in the untreated group either (P<0.05). Feiyanning Decoction is effective in inhibiting the growth of lung cancer. And the effect of inhibiting the expression of HIF-1alpha protein by Feiyanning Decoction may correlate with down-regulation of p-Akt.

Age‐dependent impairment of HIF‐1α expression in diabetic mice: Correction with electroporation‐facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells

Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) as well as its downstream target genes; and (b) could be overcome by HIF-1alpha replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells (CACs) were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1alpha gene therapy. HIF-1alpha mRNA levels in wound tissue were significantly reduced in older (4-6 months) as compared to younger (1.5-2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet-derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1alpha, followed by electroporation, induced increased levels of HIF-1alpha mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. CACs in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1alpha gene therapy corrects the age-dependent impairment of HIF-1alpha expression, angiogenic cytokine expression, and CACs that contribute to the age-dependent impairment of wound healing in db/db mice.

The Role of Hypoxia in the Differentiation of P19 Embryonal Carcinoma Cells into Dopaminergic Neurons

Nervous system development at early stage is in hypoxic environment. Very little is known about the role of hypoxia in neuronal development. P19 embryonal carcinoma (EC) cells are a widely used model for studying early neuronal development. In this study we investigated the roles of hypoxia in differentiation of dopaminergic neurons derived from P19 EC cells. Results demonstrate that hypoxia increases the percentage of differentiated neurons, especially neurons of dopaminergic phenotype. To investigate the potential mechanism involved in hypoxia promoted differentiation of dopaminergic neurons, we measured the expression of hypoxia-inducible factor 1alpha (HIF-1alpha), based on its characteristic response to hypoxia. The result shows that HIF-1alpha mRNA level in P19 EC cells increases after hypoxia treatment. It is known that HIF-1alpha regulates the expression of tyrosine hydroxylase (TH) gene through binding to its promoter. Therefore, we propose that the underlying mechanism for hypoxia promoted differentiation of dopaminergic neurons was mediated by HIF-1alpha up-regulation under hypoxia.

Hypoxia‐driven proliferation of embryonic neural stem/progenitor cells – role of hypoxia‐inducible transcription factor‐1α

We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1alpha, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O(2), 10% O(2), and 3% O(2)) for 3 days. The results showed that hypoxia, especially 10% O(2), promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1alpha mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O(2). To further elucidate the potential role of HIF-1alpha in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1alpha, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1alpha mRNA was used to knock down HIF-1alpha. We found that overexpression of HIF-1alpha caused the same proliferative effect on NPCs under 20% O(2) as under 10% O(2). In contrast, knockdown of HIF-1alpha inhibited NPC proliferation induced by 10% O(2). These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1alpha was critical in this process.

Inhibition of HIF‐1 alpha and VEGF expression by the chemopreventive bioflavonoid apigenin is accompanied by Akt inhibition in human prostate carcinoma PC3‐M cells

Progression of cancer leads to hypoxic solid tumors that mount specific cell signaling responses to low oxygen conditions. An important objective of anti-cancer therapy is the development of new drugs that suppress hypoxic responses in solid tumors. Apigenin is a natural flavone that has been shown to have chemopreventive and/or anti-cancer properties against a number of tumor types. However, the mechanisms underlying apigenin's chemopreventive properties are not yet completely understood. In this study, we have investigated the effects of apigenin on expression of hypoxia-inducible factor-1 (HIF-1) in human metastatic prostate PC3-M cancer cells. We found that hypoxia induced a time-dependent increase in the level of HIF-1alpha subunit protein in PC3-M cells, and treatment with apigenin markedly decreased HIF-1alpha expression under both normoxic and hypoxic conditions. Further, apigenin prevented the activation of the HIF-1 downstream target gene vascular endothelial growth factor (VEGF). We then showed that apigenin inhibited expression of HIF-1alpha by reducing stability of the protein as well as by reducing the level of HIF-1alpha mRNA. We also found that apigenin inhibited Akt and GSK-3beta phosphorylation in PC3-M cells. Further experiments demonstrated that constitutively active Akt blunted the effect of apigenin on HIF-1alpha expression. Taken together, our results identify apigenin as a bioflavonoid that inhibits hypoxia-activated pathways linked to cancer progression in human prostate cancer, in particular the PI3K/Akt/GSK-3 pathway. Further studies on the mechanism of action of apigenin will likely provide new insight into its applicability for pharmacologic targeting of HIF-1alpha for cancer therapeutic or chemopreventive purposes.

Down-regulation of Jab1, HIF-1α, and VEGF by Moloney murine leukemia virus-ts1infection: A possible cause of neurodegeneration

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

Up-regulation and stabilization of HIF-1α in colorectal carcinomas

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates gene expression in critical pathways involved in tumor growth and metastases. In our study we evaluated the expression of HIF-1alpha transcript and protein by quantitative real time PCR and by immunohistochemistry in a total of 78 colorectal carcinomas (CRCs) and in 8 samples of normal colorectal mucosa in order to identify the impact on prognosis of HIF-1alpha overexpression in CRCs. Our study demonstrates a significant up-regulation of HIF-1alpha mRNA as well as a high frequency of the protein immunoreactivity in colorectal cancers compared to normal samples. However, no significant correlation was found between HIF-1alpha immunohistochemical expression and any of the clinico-pathological parameters evaluated, with the only exception of the positive association between HIF-1alpha immunoreactivity and the presence of tumor necrosis. Analogously, high levels of HIF-1alpha mRNA were found to be correlated with a poor grade of tumor differentiation but no other significant association was observed. Despite the careful selection of the tumor samples, our findings do not appear to confirm, for colorectal cancers, the significant association between HIF-1alpha overexpression and tumor aggressiveness or unfavorable prognosis demonstrated for cancers of other sites. The lack of prognostic impact of HIF-1 in this site could be explained by an intricate interaction between the survival program mediated by HIF-1 and the genetic background of tumors cells in which its activation occurs.

Study on suppression of hypoxia inducible factor-1 alpha and vascular endothelial growth factor expression by siRNA targeting hypoxia inducible factor-1 alpha

To evaluate suppression efficiency of hypoxia inducible factor-1 alpha (HIF-1 alpha) specific siRNA derived from recombinant plasmid (pSUPER(H1-siHIF-1 alpha)) on both HIF-1 alpha mRNA and protein expression and concomitant downregulation of expression of downstream angiogenic factor vascular endothelial growth factor (VEGF). Stable pSUPER(H1-siHIF-1 alpha) expression cell lines were constructed by transient transfection of pSUPER(H1-siHIF-1 alpha) eukaryotic expression vector, followed by puromycin selection. Stable expression pSUPER(H1-siHIF-1 alpha) cell line with highest HIF-1 alpha inhibition efficiency determined by reverse transcription-polymerase chain reaction (RT-PCR) was cultured under normoxia (20% O(2)) and hypoxia conditions (1% O(2)) together with control cells. RT-PCR, western blot and ELISA were used to measure inhibition ability of pSUPER(H1-siHIF-1 alpha) on HIF-1 alpha and VEGF expression. Compared to the control cells, both mRNA and protein level of HIF-1 alpha and VEGF were dramatically decreased by pSUPER(H1-siHIF-1 alpha) under hypoxia conditions. Under sufficient oxygen supply situation, HIF-1 alpha mRNA level was downregulated by pSUPER(H1-siHIF-1 alpha), but pSUPER(H1-siHIF-1 alpha) did not cause suppression of VEGF expression. pSUPER(H1-siHIF-1 alpha) could decrease HIF-1 alpha expression under both normoxia and hypoxia conditions. VEGF expression was downregulated under hypoxia conditions only. Consequently, pSUPER(H1-siHIF-1 alpha) might be a powerful tool for the inhibition of retinal neovascularization.

Inhibitory effect of HCPT on expression of HIF-1α and downstream genes in hypoxic human cervical SiHa cancer cells

The hypoxic model to simulate hypoxic microenvironment in solid tumors was established and the effect of hydrocamptothecin (HCPT) on the hypoxia-induced over-expression of HIF-1alpha and VEGF genes was explored. Human cervical cancer SiHa cells were cultured in vitro under hypoxic conditions (37 degrees C, 5% CO(2), 1% O(2)) and treated with different concentrations of HCPT for 24 h. The mRNA and protein expression levels of HIF-1alpha, VEGF and Glut1 in SiHa cells were detected by semi-quantitative RT-PCR and Western blot respectively. Normoxic control groups were exposed to normoxic conditions for 24 h. Under normoxic conditions, HCPT had no obvious effects on the HIF-1alpha and VEGF gene expression. Hypoxia induced the up-regulation of HIF-1alpha protein and downstream VEGF gene, and HCPT showed a dose-dependently inhibitory effect on the hypoxia-induced over-expression of HIF-1alpha protein and VEGF gene expression in SiHa cells, whereas HCPT had no significant effect on the HIF-1alpha mRNA expression. No difference in HCPT cytotoxicity was observed between hypoxic groups and normoxic control groups. It was suggested that HCPT could inhibit the expression of HIF-1alpha protein and downstream VEGF gene in hypoxic SiHa cells in a dose-dependent manner, and the inhibitory effect was not related with HCPT cytotoxicity.

Modulation of Glucose Metabolism Inhibits Hypoxic Accumulation of Hypoxia-Inducible Factor-1α (HIF-1α)

The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect. HIF-1alpha protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O(2), 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1alpha mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR). Both inhibitors drastically reduced hypoxic HIF-1alpha accumulation (2-DG + hypoxia 2% mean HIF-1alpha protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1alpha protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1alpha mRNA levels, suggesting a translational or posttranslational mechanism. The effect of glycolysis modulation on the HIF-1alpha levels in tumor cells may provide a novel approach to therapeutically target HIF-1alpha.

The influence of different microenvironments on melanoma invasiveness and microcirculation patterns: an animal experiment study in the mouse model

To investigate the influence of different microenvironments on tumor microcirculation patterns and invasiveness capability. Melanoma B16 cells were injected into the abdominal cavity and skeletal muscle of C57 mice synchronously. CK-18 expression in melanoma was assessed to distinguish the malignant phenotype of tumors in the abdominal cavity from that in the skeletal muscle. HIF-1alpha, MMP-2 and MMP-9 expression and mRNA levels of MMP-2 and MMP-9 was detected to compare the mRNA levels of MMP-2 and MMP-9 from the two microenvironments. Cells positive for each immunohistochemical stain and the vessels representative of each type of microcirculation pattern were counted in two microenvironments. CK-18 and HIF-1alpha expression of melanoma were significantly higher in the skeletal muscle than in the abdominal cavity (P<0.05). Compared with the abdominal cavity, melanoma cells in the skeletal muscle overexpressed MMP-2 and MMP-9 (P<0.05). Real time-PCR results also showed that MMP-2 and MMP-9 mRNA levels of melanoma were higher in the skeletal muscle than in the abdominal cavity (P<0.05). VM channels and endothelium-dependent vessels were the major microcirculation pattern in the skeletal muscle and in the abdominal cavity, respectively. Different microenvironments affect invasiveness and blood supply patterns of melanoma.

Localization of immunoreactive HIF‐1α and HIF‐2α in neuroendocrine cells of both benign and malignant prostate glands

Hypoxia induces increased tumor growth by promoting angiogenic and glycolytic pathways. Tumors expressing hypoxia-inducible factor-1alpha (HIF-1alpha), an important transcriptional activator of oxygen-regulated genes, are resistant to chemotherapy and radiotherapy. The major challenge in prostate cancer therapy today is to gain a better understanding of the development of hormone-refractory tumors, which is often characterized by neuroendocrine differentiation. Here we studied the expression of HIF-1alpha and HIF-2alpha in neuroendocrine cells of the benign prostate and in prostate cancer. Tissue sections from 30 patients who underwent radical prostatectomy and from 21 patients operated by transurethral resection of the prostate were selected for immunohistochemical analysis for expression of HIF-1alpha, HIF-2alpha, androgen receptor (AR), neuroendocrine markers (chromogranin A, synaptophysin), and two gene products downstream of HIF-1alpha: VEGF and GAPDH. Immunoreactive HIF-1alpha was detected in a subpopulation of AR-negative neuroendocrine cells in benign and malignant prostate tissue. Analysis of serial sections showed that the levels of expression of GAPDH and VEGF proteins are increased in AR-negative malignant neuroendocrine cells expressing HIF-1alpha. In situ-hybridization indicated that HIF-1alpha mRNA levels are not higher in neuroendocrine prostate cancer cells relative to corresponding non-neuroendocrine tumor cells. We also demonstrated induced stabilization of nuclear HIF-1alpha in LNCaP cells by hypoxia and long-term stimulation with interleukin-6. Focal HIF-2 expression was detected in benign neuroendocrine-like cells and in malignant prostatic cells. The expression of HIF-1alpha and HIF-2alpha in prostate cancer has been confirmed, but we also identified immunoreactive HIF-1alpha and downstream gene products in benign and malignant prostate neuroendocrine cells.

Mechanism of hypoxia-induced factor 1α expression in endothelial cells of the human umbilical vein and its induction of apoptosis

Approach to mechanism of hypoxia-induced factor 1alpha expression in endothelial cells of human umbilical vein and its induction of apoptosis In vitro models, and such techniques as transmission electron microscopy, flow cytometry, RT-PCR and Western blot, were applied to investigate the transcription and protein expression of HIF-1alpha mRNA in ECV 304 cells and the action of HIF-1alpha on cell cycle blocking, proliferation inhibition and induction of apoptosis. Cells were divided into two groups: normal oxygen and hypoxic for various time periods (2, 4, 8, 12, 24 and 48 h). We observed that the expression level of HIF-1alpha mRNA and its protein were correlated with the degree of hypoxia. The expression of HIF-1alpha mRNA notably increased after 4, 8, 12, 24 and 48 h of hypoxia, particularly at 24 and 48 h with a gray scale of (71 +/- 1.81) and (70 +/- 2.02) respectively, differing significantly from the control group (P < 0.01), The protein expression of HIF-1alpha also increased 4, 8, 12, 24 and 48 h after hypoxia, particularly at 24 and 48 h, with gray scale (83 +/- 0.15) and (98 +/- 0.10) respectively (P < 0.01), indicating an increase of HIF-1alpha protein expression significantly different from the other groups (P < 0.01). In the control group, there was slight transcription and protein expression of HIF-1alpha at 0 h after hypoxia. Meanwhile, each phase of the cell cycle was detected to have increased expression of HIF-1alpha in response to oxygen-deficient treatment. At times of 24 and 48 h, the number percentage of cells in G1 significantly increased with values of 66.335 +/- 2.144 and 58.890 +/- 5.128; however, the number cells in S phase decreased to 36.215 +/- 1.582 and 39.826 +/- 5.097, significantly different compared to the control group at 0 h with values of 43.903 +/- 6.506 and 60.571 +/- 24.026-(P < 0.05). When the cell cycle was blocked in the G2/S phase, the cell apoptosis ratio significantly increased by 9.24 +/- 1.828 and 30.735 +/- 11.38, compared to each control group-(P < 0.01). By induction of hypoxia, the cell cycle was dramatically blocked in G1/S phase, and the expression of HIF-1alpha also increased Therefore, sustained expression of HIF-1alpha can inhibit cell hyperplasia, and the apoptosis promotion of injured cells as well as provide protection of endothelial cells.

Aberrant Expression of Leptin in Human Endometriotic Stromal Cells Is Induced by Elevated Levels of Hypoxia Inducible Factor-1α

Elevated expression of leptin in endometriotic tissue results in an increase in stromal cell proliferation and may contribute to the development of endometriosis. However, the underlying mechanism responsible for aberrant expression of leptin is not known. We hypothesize that aberrant expression of leptin in endometriotic stroma may be regulated by increased levels of hypoxia-inducible factor-1alpha (HIF-1alpha), the master transcription factor that controls gene expression in response to hypoxia. Herein we show that the mRNA and protein levels of HIF-1alpha were greater in ectopic endometriotic tissue compared with its eutopic counterpart. Exposure of eutopic endometrial stromal cells under hypoxic conditions or treated with desferrioxamine (DFO, chemical hypoxia) resulted in a time-dependent increase in leptin gene expression. A promoter activity assay demonstrated that HIF-1alpha induced leptin promoter activity after DFO treatment. Chromatin immunoprecipitation assay further demonstrated that binding of HIF-1alpha to leptin promoter was evident after DFO treatment. Finally, depletion of HIF-1alpha by short interference RNA abolished leptin expression in ectopic endometriotic stromal cells. Taken together, our data demonstrate that aberrant expression of leptin in ectopic endometriotic stromal cells is induced, at least in part, by an elevated level of HIF-1alpha in these cells, providing new insights into the etiology of endometriosis.