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.

OBJECTIVE

To assess the expression of connective tissue growth factor (CTGF), and relevant mechanism for the regulation of CTGF expression by hypoxia in human renal interstitial fibroblast.

METHODS

A human renal interstitial fibroblast cell line TK173 was treated under hypoxia (1% O(2)) or nomoxia (21% O(2)) condition. The expressions of HIF1-alpha, hypoxia marker protein, and CTGF protein were analyzed by Western blotting. RT-PCR was carried out to measure the levels of CTGF mRNA. The activations of MAPKs (ERK, JNK, p38) signaling pathways were assessed at different time points (30 min, 1 h, 6 h, and 12 h ), and the changes of CTGF expression were detected after the inhibitors of activation of MAPKs were applied, respectively.

RESULTS

The expression of HIF-1alpha protein appeared in cells under hypoxia for 6 h. The expressions of CTGF protein were up-regulated in TK173 cells under hypoxia for 12 h, reached the peak levels in 2 folds of normoxia group cells for 24 h, and return to the levels of control cells by 48 h. The levels of CTGF mRNA were elevated in cells under hypoxia for 1 h, significantly increased at 6 h (6.6+/-1.0, P=0.000 2), and returned to the levels of normoxia group cells by 24 h. Activations of ERK1/2, JNK and p38 were seen in hypoxic cells. Activation of ERK1/2 and JNK were occurred as early as at 10 min, and reached the peak levels at 1 h, while the peak levels of activated JNK were seen at 30 min, then the levels of activated ERK1/2, p38, and JNK were all declined at 6 h, back to the baseline levels at 12 h. Blockade of ERK activation with PD98059, and blockade of JNK activation with SP600125 did not suppress hypoxia-induced expression of CTGF protein, whereas blockade of p38 MARK activation with SB203580 abolished hypoxia-induced expressions of CTGF protein and CTGF mRNA.

CONCLUSIONS

Hypoxia could stimulate the expression of CTGF in human renal interstitial fibroblast through the activation of p38 MARK signaling pathway.

OBJECTIVE

Different studies have linked hypoxia to embryonic development. Specifically, when embryonic pancreases are cultured ex vivo under hypoxic conditions (3% O2), beta cell development is impaired. Different cellular signalling pathways are involved in adaptation to hypoxia, including the ubiquitous hypoxia-inducible-factor 1-α (HIF1-α) pathway. We aimed to analyse the effects of HIF1-α stabilisation on fetal pancreas development in vivo.

METHODS

We deleted the Vhl gene, which encodes von Hippel-Lindau protein (pVHL), a factor necessary for HIF1-α degradation, by crossing Vhl-floxed mice with Sox9-Cre mice.

RESULTS

HIF1-α was stabilised in pancreatic progenitor cells in which the HIF pathway was induced. The number of neurogenin-3 (NGN3)-expressing cells was reduced and consequently endocrine development was altered in Vhl knockout pancreases. HIF1-α stabilisation induced Vegfa upregulation, leading to increased vascularisation. To investigate the impact of increased vascularisation on NGN3 expression, we used a bioassay in which Vhl mutant pancreases were cultured with or without vascular endothelial growth factor (VEGF) receptor 2 (VEGF-R2) inhibitors (e.g. Ki8751). Ex vivo analysis showed that Vhl knockout pancreases developed fewer NGN3-positive cells compared with controls. Interestingly, this effect was blocked when vascularisation was inhibited in the presence of VEGF-R2 inhibitors.

CONCLUSIONS

Our data demonstrate that HIF1-α negatively controls beta cell differentiation in vivo by regulating NGN3 expression, and that this effect is mediated by signals from blood vessels.

Hypoxia serves a critical role in the pathogenesis of liver fibrosis. Hypoxia‑inducible factor 1α (HIF1‑α) is induced when cells are exposed to low O2 concentrations. Recently, it has been suggested that Rho‑associated coiled‑coil‑forming kinase 1 (ROCK1) may be an important HIF1‑α regulator. In the present study, it was analyzed whether crosstalk between HIF1‑α and ROCK1 regulates cell proliferation and collagen synthesis in hepatic stellate cells (HSCs) under hypoxic conditions. For this purpose, a rat hepatic HSC line (HSC‑T6) was cultured under hypoxic or normoxic conditions, and HIF1‑α and ROCK1 expression was measured at different time points. Additionally, HSC‑T6 cells were transfected with HIF1‑α small interfering RNA (siHIF1‑α), and measured protein expression and mRNA transcript levels of α‑smooth muscle actin, collagen 1A1 and ROCK1. Collagen 3A1 secretion was also measured by ELISA. Cell proliferation was assessed by the MTT assay under these hypoxic conditions. The results indicated that a specific ROCK inhibitor, Y‑27632, increased HIF1‑α and ROCK1 expression over time in HSC‑T6 cells in response to hypoxia. In addition, knockdown of HIF1‑α inhibited HSC‑T6 proliferation, suppressed collagen 1A1 expression, decreased collagen 3A1 secretion and attenuated ROCK1 expression. Notably, ROCK1 inhibition caused HSC‑T6 quiescence, suppressed collagen secretion and downregulated HIF1‑α expression. Collectively, these findings indicated that the interplay between HIF1‑α and ROCK1 may be a critical factor that regulates cell proliferation and collagen synthesis in rat HSCs under hypoxia.

Hypercarbia, or elevated carbon dioxide, is an environmental challenge that can have detrimental effects on the physiology and performance of aquatic organisms. With aquatic hypercarbia predicted to become more prevalent in the future due to global climate change, it is important to quantify how hypercarbia impacts aquatic organisms, especially fish. The impact of hypercarbia on the behavior and physiology of fishes has been well studied, but relatively few studies have examined the molecular processes that underlie resulting behavioral and physiological changes. In an effort to define the molecular response of fishes to acute hypercarbia exposure, bluegill (Lepomis macrochirus) and silver carp (Hypophthalmichthys molitrix) were exposed to either 30 mg L(-1) CO2 (pCO2≈15,700 μatm) or ambient (10 mg L(-1) CO2; pCO2≈920 μatm) conditions for 1h and the expression of a variety of genes, across three tissues, were compared. Exposure to 30 mg L(-1) CO2 in bluegill and silver carp resulted in an increase in c-fos, hif1-α, and gr-2 transcripts, while silver carp alone showed increases in hsp70 and hsc70-2 mRNA. This study demonstrates that acute hypercarbia exposure impacts gene expression in a species and tissue specific manner, which can be useful in identifying potential mechanisms for hypercarbia tolerance between species, and pinpoint specific tissues that are sensitive to hypercarbia exposure.

Kisspeptin is a peptide encoded by the Kiss 1 gene and is also called metastin. Previous studies have generally focused on several functions of this peptide, including metastasis, puberty, vasoconstriction and reproduction. However, few studies have focused on the cardiac functions of kisspeptin. In the present study, cardiac histomorphology was observed via TEM (transmission electron microscope) and HE and Masson staining to observe instinctive changes. Serum metabolites levels were also measured and analyzed using GC/TOF-MS after injection with kisspeptin-10. A gene chip was employed to screen the potential genes and pathways in the myocardium at the transcriptional leve, while RT-PCR and Western Blot were conducted to verify the relevant mRNA and protein expression, respectively. Histopathological findings demonstrated that there were many irregular wavy contractions through HE staining and increased fibrosis around the heart cells through Masson staining after treatment with kisspeptin-10. Additionally, the main changes in ultrastructure, including changes in mitochondrial and broken mitochondrial cristae, could be observed with TEM after treatment with kisspeptin-10. The PCA scores plot of the serum metabolites was in the apparent partition after injection of kisspeptin-10. Twenty-six obviously changed metabolites were detected and classified as amino acids, carbohydrate metabolites, organic acids and other metabolites. Furthermore, gene chip analysis showed 1112 differentially expressed genes after treatment with kisspeptin-10, including 330 up-regulated genes and 782 down-regulated genes. These genes were enriched in several signaling pathways related to heart diseases. The RT-PCR result for ITGB8, ITGA4, ITGB7, MYL7, HIF1-α and BNP corresponded with the gene chip assay. Moreover, the upregulated genes ITGB8, ITGA4 and BNP also displayed consistent protein levels in Western Blot results. In summary, these findings suggest that kisspeptin-10 could alter the morphology and structure of myocardial cells, serum metabolite levels, and expression of genes and proteins in heart tissues. Our work determined the profound effects of kisspeptin-10 on the heart, which could further lead to the development of therapeutics related to kisspeptin-10, including antagonists and analogs.

The objective of this work is to study the protective effects of Quercetin against sodium nitrite-induced hypoxia on liver, lung, kidney and cardiac tissues, also to explore novel mechanism of this compound. Male albino rats were injected with sodium nitrite (75 mg/kg). Quercetin (200 mg kg-1 ,- i.p.) was administrated 24 and 1 h respectively prior to sodium nitrite intoxication, hypoxia significantly decreased hemoglobin concentration, while increased expressions of HIF, Bax, Smad-2, TGF-β, and AKT. However, administration of Quercetin played a modulatory role against the previous mentioned apoptotic factors protein expressions in all the studied tissues. On the other hand, Bcl-2 was downregulated by NaNO2 , whereas concurrent treatment with Quercetin increased its expression. It was concluded that Quercetin possesses an anti-apoptotic action induced by NaNO2 -intoxication via different mechanisms. Quercetin administration is recommended in areas of high altitudes to combat the hazard effect of hypoxia in different organs and in some diseases accompanied by hypoxic stress.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer characterized by a high degree of recurrence, angiogenesis, and metastasis. The importance of alternative pro-angiogenesis pathways including viral factors has emerged after decades of directly targeting various signaling components. Using NPC as a model, we identified an essential oncogenic pathway underlying angiogenesis regulation that involves the inhibition of a tumor suppressor, Spry3, and its downstream targets by EBV-miR-BART10-5p (BART10-5p) and hsa-miR-18a (miR-18a). Overexpression of EBV-miR-BART10-5p and hsa-miR-18a strongly promotes angiogenesis in vitro and in vivo by regulating the expression of VEGF and HIF1-α in a Spry3-dependent manner. In vitro or in vivo treatment with iRGD-tagged exosomes containing antagomiR-BART10-5p and antagomiR-18a preferentially suppressed the angiogenesis and growth of NPC. Our findings first highlight the role of EBV-miR-BART10-5p and oncogenic hsa-miR-18a in NPC angiogenesis and also shed new insights into the clinical intervention and therapeutic strategies for nasopharyngeal carcinoma and other virus-associated tumors.

Keywords: antiangiogenesis; exosome; miRNA; nasopharyngeal carcinoma.

Over the past decade evidence from multiple research trajectories have converged to provide evidence that impaired glucose metabolism and mitochondrial dysfunction in the brain are the critical issues laying at the root of Bipolar Disorder (BD). These developments have been paralleled by increasing recognition of the systemic metabolic dysfunction accompanying mood disorders. Significant insulin resistance (IR) occurs in BD patients and this has been demonstrated to be related to illness severity independent of medication status. Preliminary evidence for a therapeutic effect of a Ketogenic Diet (KD) in BD and other neuropsychiatric conditions has recently refocused interest in the role of IR in BD pathogenesis. In this paper we review evidence of hyperinsulinemia in BD as the primary cause of mitochondrial dysfunction mediated by impairment of the PI3K/AKT/HIF1-a insulin signaling pathway. This cascade of dysfunction directly suppresses the Pyruvate Dehydrogenase Complex through HIF1-a mediated activation of Pyruvate dehydrogenase kinase 1 (PDK1) leading to the Warburg effect and mitochondrial dysfunction. We review evidence that the KD acts directly on each of these mechanisms and propose that a trial of KD in BD with a mechanistic component is needed to further investigate the role of IR in BD.

OBJECTIVE

To investigate the expression of HIF1-alpha in heart and lung tissue died from asphyxia.

METHODS

The rats model of asphyxia death was constructed by hanging, different asphyxia groups and control group sets were made according the postmortem time (0,2,6,24 h), immunohistochemistry and half-quantitative RT-PCR methods were used to investigate expression of HIF1-alpha and mRNA changes on heart and lung tissue.

RESULTS

The positive staining of HIF1-alpha could be observed in the myocardium and lung tissue. Significant differences were found between the groups of asphyxia and their corresponding control group. HIF1-alpha expression was found in all the asphyxia groups while it was only expressed in the control groups of 2 h, 6 h and 24 h. Nucleic positive staining could be detected in all the asphyxia groups but none was found in the control groups. RT-PCR showed that the expression of mRNA between 0 h asphyxia group and 0 h control group were equal in both cardic muscle and lung, but elevated expression in groups of 2,6,24h compared to their control groups.

CONCLUSIONS

The nuclear positive staining of HIF1-alpha in heart and lung can be a special character of suffocation death.

BACKGROUND

Surgical weight loss response is variable, with suboptimal outcomes in some patients. We hypothesized that genetic biomarkers may be related to weight change.

METHODS

We tested 330 single nucleotide polymorphisms (SNPs) in genes relevant to metabolic regulation in 161 patients whose decrease in body mass index (BMI), 1 year after laparoscopic adjustable gastric banding (LAGB) or Roux-en-Y gastric bypass (RYGB), was small (lowest quartile response) or large (highest quartile response). LAGB patients whose BMI decreased≤4.7 or≥10.2 units comprised groups I (n = 43) and II (n = 40), respectively. RYGB patients whose BMI decreased≤13.6 or≥19.8 units comprised groups III (n = 39) and IV (n = 39), respectively. Within each surgery, SNPs with large differences in reference allele frequency (z score>2, corresponding to values displaced 2 standard deviations [SD] from the mean for all SNPs) in low versus high quartiles, were identified. We compared reference allele frequencies, within surgical procedure, using the χ(2) test (using Bonferroni correction for multiple testing).

RESULTS

The mean percent excess weight losses (±SD) corresponding to groups I, II, III, and IV were: 16 (±12), 64 (±30), 55 (±16), and 75 (±17), respectively. SNPs with z score>2 were identified in genes involved in LAGB response, lipid metabolic regulation (APOE, rs439401; APOC4, rs2288911), neural processes (DRD3, rs167771; HTR3 B, rs3758987), and xeno- or endobiotic metabolism (CYP3 AARB1, rs10846744), folate metabolism (MTHFR, rs2066470), regulation of glycolysis in immune cells (HIF1 A, rs1951795), vitamin K cycling (VKORC1, rs2359612), and xeno- or endobiotic metabolism (CYP3 AAGB response, APOE SNP frequencies were significantly different.

CONCLUSIONS

With further validation, information derived from patient DNA may be useful to predict surgical weight loss outcomes and guide selection of surgical approach.

One limitation of gene-directed enzyme prodrug therapy (GDEPT) is the difficulty in selectively and efficiently transducing cancer cells with the gene encoding a prodrug-converting enzyme. To circumvent this issue, we sought to move the selectivity from the gene delivery level to the protein level. We developed fusion proteins of the prodrug-activating enzyme yeast cytosine deaminase (yCD) and the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain that regulates the accumulation of HIF-1α in an oxygen-dependent manner. We called these HOPE fusions for HIF1-α Oxygen-dependent degradation domain/Prodrug-converting Enzyme. The HOPE fusions were designed to selectively accumulate in cells experiencing hypoxia and thus selectively cause conversion of the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil (5-FU) where oxygen levels are low (e.g., at the center of a tumor). Consistent with our hypothesis, HT1080 fibrosarcoma cells transduced with HOPE fusion genes exhibited increased fusion protein accumulation and increased sensitization to 5-FC in mock-hypoxia.

OBJECTIVE

HIF is an important transcription-regulator for adaptation to cellular stress in cells of myeloid origin. Classically, expression and activity of HIF1-α is regulated by oxygen-concentration within cell. However, there exists an alternative regulatory mechanism affecting HIF1-α levels independent of oxygen concentration particularly in inflammatory cells like macrophages. Here we report the mechanism of HIF1-α upregulation in TAMs by Oncostatin-M (OSM) independent of cellular oxygen concentration.

METHODS

THP-1 derived macrophages were treated with OSM. HIF1-α levels and interaction with pVHL were evaluated via immunoblot-analysis and Co-immunoprecipitation. Translocation of HIF1-α to nucleus was visualized using confocal-microscopy. Fold change in mRNA levels of ARG-1 and COX-2 was analyzed using RT-PCR.

RESULTS

Current study demonstrates that OSM treatment to TAMs led to an increased expression of HIF1-α under normoxic conditions via activation of mTORC2. This HIF1-α upregulation was dependent on both de novo synthesis of HIF1-α and its enhanced stability due to disruption of its binding to pVHL. Furthermore, we evaluated that OSM not only enhances the expression of HIF1-α but also increases its localization to nucleus where it acts as a transcription factor regulating expression of genes like ARG-1 and COX-2.

CONCLUSIONS

Inflammation is a critical hallmark of cancer as tumor microenvironment is largely infiltrated with macrophages. These tumor associated macrophages (TAMs) display a M2 skewed phenotype. Many target genes of TAMs are HIF1-α responsive. These TAMs are involved in tumor progression, metastasis and angiogenesis. Targeting of HIF1-α/OSM can lead to devising of better therapeutic strategy against cancer.

OBJECTIVE

To identify and quantitate hypoxia inducible factor 1 alpha (HIF1 α) isoforms in circulating peripheral blood mononuclear cells (PBMCs), and to assess their effects on target gene expression in rheumatoid arthritis (RA) patients.

METHODS

PBMCs from healthy controls and from RA patients were analysed ex-vivo for expression of HIF isoforms, and target genes were assessed by RT-PCR.

RESULTS

Transcripts of multiple HIF1α isoforms exist in circulating PBMCs. Expression of all these isoforms is dramatically, and maximally, augmented by foreign surface recognition. However, HIF1α protein stabilisation requires additional cell activation with phorbol ester. No difference in the expression or regulation of the HIF1α isoforms was seen between patients with active RA and healthy controls. However, analysis of a panel of HIF1α target genes revealed increased basal expression of the adrenomedullin gene in RA PBMCs, with resulting loss of further induction upon cell activation.

CONCLUSIONS

Even in normoxia PBMCs express stable HIF1α protein on cell activation. Whilst multiple HIF1α isoforms exist in PBMCs no differences in expression were seen in RA compared with healthy controls. RA causes constitutive adrenomedullin expression in PBMCs that is not explicable by altered HIF expression, or stabilisation. Adrenomedullin has a variety of potential biological roles in RA, including regulation of angiogenesis, and aberrant gene regulation may be relevant in RA pathogenesis.

This study examined the effects of fucoidan on epithelial-to-mesenchymal transition (EMT) in a human triple-negative breast cancer (TNBC) cell line in a hypoxic microenvironment. Transwell and wound-healing assays were performed to analyze the invasion and migration of MDA-MB-231 human mammary cancer cells, respectively. The expression levels of EMT markers and hypoxia-inducible factor-1α (HIF-1α) were detected through western blotting. Under hypoxia, fucoidan treatment inhibited proliferation of breast cancer cells. Fucoidan also suppressed the invasion and migration of MDA-MB-231 cells. Western blotting revealed that fucoidan treatment significantly reduced the protein expression levels of HIF-1α and HIF-1 target genes. Furthermore, the nuclear translocation and activity of HIF-1α were reduced. Fucoidan treatment significantly downregulated the expression levels of mesenchymal markers (N-cadherin and vimentin), but upregulated the expression levels of the epithelial markers zonula occludens-1 and E-cadherin. In addition, overexpression of HIF1-α protected cells from fucoidan-mediated suppression of migration and invasion. These data suggested that fucoidan may inhibit EMT in human TNBC cells via downregulation of the HIF1-α signaling pathway.

A better understanding of the factors governing the vascularization of engineered tissues is crucial for their advancement as therapeutic platforms. Here, we studied the effect of implant volume and cell densities on the in vivo vascularization of modular engineered tissue constructs. Sub-millimeter collagen modules containing adipose-derived mesenchymal stromal cells (adMSC) and enveloped by human umbilical vein endothelial cells (HUVEC) were subcutaneously implanted in severe-combined immunodeficient mice with a beige-mutation (SCID-bg) mice. Implant volume and cell density was varied relative to a base case, defined as a 0.01 mL implant containing 1.5×10(7) adMSC/mL and 3.9×10(6) HUVEC/mL. At 7 and 14 days post-transplantation, the constructs were harvested for immunohistochemical analysis of total (CD31(+)) and graft-derived (UEA1(+)) vessel formation, hypoxia-inducible factor 1-alpha (HIF1α) expression, infiltration of host-derived leukocytes (CD45), and macrophages (F4/80). Implant volume and cell density affected the relative contributions of host- versus graft-derived vascularization, highlighting that different mechanisms underlie the two processes. Graft-derived vessel formation was most rapid and robust in implants with high HIF1α expression, namely large volume implants and implants with high adMSC and HUVEC density (p<0.01 compared to base case at day 7). Many HIF1α(+) cells were vessel-lining HUVEC, suggesting that HIF1 activation may be key to vessel assembly in the graft. Host vessel ingrowth, however, dominated the vascularization of small volume implants (of high and low adMSC density alike), which showed low HIF1α expression at day 7. Host vessels were sustained to day 14 when adMSC density alone was increased, presumably due to increased paracrine secretions. This study points to a potential role of HIF1 activation in the vascularization of tissue constructs, which may be harnessed to engineer robust vessels for therapeutic applications.

The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 μmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 μmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 μmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 μmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 μmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 μmol/L)-treated group or between DMSO-treated control group and gefitinib (1 μmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 μmol/L), and NVP-BKM120 (1 μmol/L) or NVP-BKM120 (1 μmol/L) plus gefitinib (1 μmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 μmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.

OBJECTIVE

To investigate whether anemia, a putative factor counteracting the efficacy of radiotherapy, up-regulates the endogenous markers of intratumoral hypoxia, the hypoxia inducible factors HIF1 alpha and HIF2 alpha.

METHODS

Forty-two hysterectomy specimens harboring endometrial adenocarcinomas of the endometrioid cell type, stage I were investigated immunohistochemically for the expression of HIF1 alpha, HIF2 alpha and the down-stream inducible expression of vascular endothelial growth factor VEGF. The results were correlated with hemoglobin (Hb) levels.

RESULTS

There was no significant association between Hb levels and the expression of HIF1 alpha, HIF2 alpha or VEGF in our material.

CONCLUSIONS

Activation of hypoxia pathways is an intrinsic self-regulated process, independent of Hb levels in endometrial adenocarcinomas. Other factors such as microvessel density (MVD), vessel/tumor cell distance or genetic events may be important.

OBJECTIVE

To investigate the expression and significance of HIF1 alpha in hepatocellular carcinoma (HCC) tissues and in hepatoma carcinoma cell line HepG2.

METHODS

The expression of the HIF1 alpha mRNA and protein were detected with immunohistochemistry (IHC), Western blot and RT-PCR techniques in HCC, normal liver tissues and HepG2. Their relationship with the pathological characteristics of the HCC was also analyzed.

RESULTS

HIF1 alpha protein was obviously expressed in HCC. The positive rate of HIF1 alpha protein in HCC tissues was 76.4% and was higher than that in normal hepatic tissues. The expression of HIF1 alpha had a correlation to the differentiation degree of HCC tissues and intrahepatic and extrahepatic metastases (P<0.05), but there was no correlation to the existence of portal vein tumor emboli, the status of HBsAg and the prognosis (P<0.05). The results of Western blot and RT-PCR were similar to the results of IHC. The positive rate of HIF1 alpha in HepG2 was 93.6%. The levels of HIF1 alpha protein and mRNA began to increase after being treated two hours with hypoxia or with CoCl(2) (150 micromol/L).

CONCLUSIONS

HIF1 alpha protein is obviously expressed in HCC and it is mainly affected by hypoxia. The expression of HIF1 alpha is related to the differentiation of the HCC and its intrahepatic and extrahepatic metastases but has no correlation to the existence of portal vein tumor emboli, the status of HBsAg and the prognosis.

We evaluated the role of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and hypoxia-inducible factor 1-a (HIF1-a) in melanoma angiogenesis and investigated their expression in dysplastic nevi, as potential melanoma precursors. In addition, we examined a possible correlation of VEGF expression with PlGF and HIF1-a. These factors were detected immunohistochemically in 95 melanomas of all types and stages and in 28 dysplastic nevi. We used 10 intradermal melanocytic nevi as controls. HIF1-a was expressed in 93 out of 95 (97.89%) melanomas and in none of the dysplastic or control nevi. HIF1-a expression was more intense in melanocytes around necrotic areas but did not correlate with melanoma type, the Clark staging or the Breslow thickness. A strong positive association was detected between HIF1-a and VEGF expression in all cases. VEGF was detected in 82 out of 95 (86.31%) melanomas and in 21 out of 28 (75%) dysplastic nevi, whereas it was expressed weakly in neoplastic cells of the controls. Its expression was more intense in melanomas, especially in nodular melanomas of elevated stage and thickness. PIGF was detected in 46 out of 95 (48.42%) melanomas and in none of the nevi. Expression did not correlate with melanoma staging nor thickness; however, it was more intense in superficial spreading melanomas, where a weak positive association between VEGF and PlGF was also detected. There was no association between HIF1-a and PlGF in any melanoma type. Hypoxia, through the expression of HIF1-a, plays a key role in melanoma progression; it activates VEGF secretion, which induces angiogenesis and metastasis. The role of PlGF seems to be limited.

Signal transducer and activator of transcription 3 (STAT3) interacts with many gene promoters and transcription factors such as hypoxia-induced factor 1α (HIF-1α). Recent evidences proposed that STAT3 and HIF-1α together are responsible for angiogenesis and immune response suppression. The main aim of this study was to inhibit STAT3 and HIF-1α and assess their effects on the expression of immunosuppressive cytokines. S31-201 and PX-478 were used to inhibit STAT3 and HIF-1α, respectively. In both hypoxic and normoxic conditions, intracellular levels of HIF-1α were evaluated by western blotting and flow cytometry. Supernatant levels were also measured for VEGF, IL-10, and TGF-β concentration. S31-201 suppressed proliferation of MCF-7 cells and led to reduced HIF-1α expression in both hypoxic and normoxic conditions. It also decreased production of the immunosuppressive cytokines. STAT3 inhibition suppressed tumor cell growth and cytokine production in a HIF-1α-dependent manner, and can be used as a promising target in cancer therapies.

Due to the low number of collectable stem cells from single umbilical cord blood (UCB) unit, their initial uses were limited to pediatric therapies. Clinical applications of UCB hematopoietic stem and progenitor cells (HSPCs) would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity. In recent years, numerous attempts have been made to expand human UCB HSPCs in vitro. In this study, we report that caffeic acid phenethyl ester (CAPE), a small molecule from honeybee extract, can promote in vitro expansion of HSPCs. Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells. Importantly, culture of CD34(+) HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units. Burst-forming unit-erythroid was the mostly affected colony type, which increased more than 3.7-fold in 1 μg mL(-1) CAPE treatment group when compared to the controls. CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α. Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.

We investigated whether intrapancreatic coagulation, with deposition of the fibrinogen-γ dimer (Fib-γD) and hypoxia, affect the severity of acute pancreatitis (AP) in mice. Pancreata of mice with AP induced by administration of cerulein or by L-arginine, or from patients with pancreatitis, had increased deposition of Fib-γD compared with control pancreata. Heparin administration protected mice from cerulein-induced AP and prevented Fib-γD formation. Cerulein administration resulted in activation and stabilization of hypoxia-inducible factor-1α (HIF1α) in pancreata of oxygen-dependent degradation domain-luciferase HIF1α reporter mice. Cerulein also led to induction of genes regulated by HIF1α, including Vegfa and Ero1a, before evidence of Fib-γD deposition or histologic features of AP. Expression of tissue factor, which is regulated by vascular endothelial growth factor, also increased following cerulein administration. Mice with acinar cell-specific disruption of Hif1a (Hif1aAc-/-) developed spontaneous endoplasmic reticulum stress and less severe AP, but did not accumulate Fib-γD following administration of cerulein. Feeding mice increased pancreatic expression of HIF1α, indicating a physiologic role in the exocrine pancreas. Therefore, HIF1α has bifunctional roles, in exocrine pancreas homeostasis and progression of AP that is promoted by intrapancreatic coagulation.

The aim of the current study is to evaluate the efficacy of pretreatment with either l-arginine (L-arg) or Carnosine (Car) and their combination in ameliorating some of the biochemical indices induced in the lung of sodium nitrite (NaNO2 )-intoxicated rats. The results revealed that NaNO2 significantly increased serum tumor necrosis factor-α, C-reactive protein, heat shock proteins-70, vascular endothelial growth factor, and Interleukin 6. Moreover, transforming growth factor-β, hypoxia-inducible factor, Smad-2, Protein Kinase B (AKT), and Bax were overexpressed, whereas Bcl2 protein was downregulated compared with the normoxic group. The administration of the fore mentioned antioxidants, either alone or in combination, markedly downregulated the previously mentioned inflammatory, apoptotic, as well as the fibrotic markers in lung tissue compared with the NaNO2 -intoxicated rats. The histopathological examination reinforced the previous results. In conclusion, the current data revealed the efficacy of l-arg and Car in ameliorating the pulmonary damage via suppression of the inflammatory markers in response to NaNO2 -intoxication. Interestingly the combination regimen showed the most significant effect.