Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.     

Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α in asthenozoospermia

Impaired male fertility may have a variety of causes, among which asthenozoospermia. In its etiology, several bioactive substances, such as cytokines may be involved. In this context, our aim was to evaluate the expression of interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α, in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. We evaluated twenty-eight infertile patients affected by idiopathic asthenozoospermia and twenty-three normospermic fertile donors, age-matched. Sperm parameters were evaluated; immunohistochemical analysis and enzyme-linked immunosorbent assay were then performed in isolated spermatozoa. Spermatozoa from the asthenozoospermic group presented an increased expression of IL-1β, COX-2, and HIF-1α compared with the normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence sperm motility. Nevertheless, further studies are needed in order to assess whether these bioactive mediators have a potential relevance as targets in future therapeutic strategies for the treatment of male infertility.     

Mitochondrial reactive oxygen species mediates nicotine-induced hypoxia-inducible factor-1α expression in human non-small cell lung cancer cells

Cigarette smoking is not only a documented risk for lung carcinogenesis but also promotes lung cancer development. Nicotine, a major component of cigarette smoke but not a carcinogen by itself, has been found to induce proliferation, invasion and metastasis of non-small cell lung cancer (NSCLC). Here we reported that proinvasive effect of nicotine is analogous to that of hypoxia and involves stabilization and activation of hypoxia-inducible factor (HIF)-1α, a key factor in determining the presence of HIF-1 and expression of its downstream metastasis-associated genes. Furthermore, nicotine-induced upregulation of HIF-1α was dependent on mitochondria-derived reactive oxygen species (ROS). Ecotopic expression of mitochondrial targeted catalase effectively prevented nicotine-induced accumulation of HIF-1α protein. In addition, we demonstrated that the effect of nicotine in upregulation of HIF-1α was mediated by Dihydro-β-erythroidine (DhβE)-sensitive nicotine acetylcholine receptors (nAChRs) and required synergistic cooperation of Akt and mitogen-activated protein kinase (MAPK) pathways. These results suggest that exposure to nicotine could mimic effects of hypoxia to stimulate HIF-1α accumulation and activity that might underlie the high metastatic potential of lung cancer.     

Expression of hypoxia-inducible factor-1α and vascular density in mammary adenomas and adenocarcinomas in bitches

Background

The study aimed at examining hypoxia-inducible factor (HIF)1α expression in adenocarcinomas and adenomas in bitches in regard to tumour malignancy grade, proliferation, apoptosis and vascularisation. Therefore, paraffin sections of 15 adenomas and 64 adenocarcinomas sampled from 79 dogs aged 6 to 16 years were analysed.

Results

A significantly higher HIF-1α expression was noted in adenocarcinomas in comparison to adenomas (P?<?0.0004). Moreover, HIF-1α expression in adenocarcinomas correlated positively with tumour malignancy grade (r?=?0.59, P?<?0.05), Ki-67 antigen expression (r?=?0.43; P?<?0.0005), TUNEL-positive cells (r?=?0.62, P?<?0001) and tumour vascularity measured by quantification of vessels characterized by the expression of von Willebrand Factor (r?=?0.57, P?<?0.05).

Conclusion

Results of this study indicate a similar biological role of HIF-1α in dogs and in humans, which may confirm suitability of the animal model in investigations on progression of tumours in humans.

     

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.     

Up-regulation of hypoxia-inducible factor-1α enhanced the cardioprotective effects of ischemic postconditioning in hyperlipidemic rats

Hyperlipidemia is an independent risk factor in the devel- opment of ischemic heart disease, which can increase myo- cardial susceptibility to ischemia/reperfusion （I/R） injury. Ischemic postconditioning （PostC） has now been demon- strated as a novel strategy to harness nature＇s protection against myocardial I/R injury in normal conditions. However, the effect of PostC on hyperlipidemic animals remains elusive. It has been shown in our previous study that PostC reduces the myocardial I/R injury, and hypoxia- inducible factor-1α （HIF-1α） may play an important role in the cardioprotective mechanisms of PostC on normal rats. Here, we tested the hypothesis that the cardioprotec- tion of PostC on hyperlipidemic rats is associated with the up-regulated HIF-1α expression. Male Wistar rats were fed with a high-fat diet for 8 weeks, and then randomly div- ided into five groups： sham, I/R, dimethyloxalylglycine （DMOG） ＋ I/R, PostC, and DMOG ＋ PostC group. The detrimental indices induced by I/R injury included infarct size, plasma creatine kinase （CK） activity and caspase-3 ac- tivity. The results showed that PostC could reduce the infarct size, when compared with the I/R group, which was consistent with the significant lower levels of plasma CK activity and caspase-3 activity, and that it increased the ex- pression of HIF-1α in hyperlipidemic rats. When DMOG was given before PostC to up-regulate HIF-1α protein level, the degree of I/R injury was attenuated. In conclu- sion, these data suggested that the up-regulation of HIF-1α may be one of the cardioprotective mechanisms of PostC against I/R injury in hyperlipidemic rats.     

Vasohibin induces prolyl hydroxylase-mediated degradation of hypoxia-inducible factor-1α in human umbilical vein endothelial cells

Vasohibin is thought to be an important negative feedback regulator of angiogenesis that is selectively induced in endothelial cells by VEGF. Here, we assessed the role of vasohibin on HIF-1α expression under oxidative stress induced by hydrogen peroxide (H₂O₂) in HUVEC. VEGF induced significant cell growth that was associated with an increase in vasohibin expression. Following H₂O₂-pretreatment, VEGF further increased cell growth but this was contrastingly associated with a decrease in vasohibin expression when compared with VEGF alone. Interestingly, vasohibin inhibited cell proliferation through degradation of HIF-1α expression during H₂O₂-pretreatment. Furthermore, vasohibin elevated the expression of prolyl hydroxylase (PHD). These results suggest that vasohibin plays crucial roles as a negative feedback regulator of angiogenesis through HIF-1α degradation via PHD.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

Chronic hypoxia- and monocrotaline-induced elevation of hypoxia-inducible factor-1α levels and pulmonary hypertension

A close relationship exists between hypoxia-inducible factor (HIF)-1 and pulmonary hypertension. The present study was carried out to explore if there are temporal alterations in HIF-1 levels during prolonged hypoxia and after monocrotaline (MCT) treatment. First, young Wistar rats were divided into 5 groups: control, hypoxia-1, hypoxia-2, hypoxia-3 and hypoxia-4. Hypoxic rats were placed in a closed hypobaric chamber (380 mm Hg) for a 1-week (hypoxia-1), 2-week (hypoxia-2), 3-week (hypoxia-3) or 5-week (hypoxia-4) period. Second, other young Wistar rats were divided into 4 groups: control, MCT-1, MCT-2 and MCT-3. MCT-treated rats were injected subcutaneously once with MCT (60 mg/kg) for a 1-week (MCT-1), 2-week (MCT-2) or 3-week (MCT-3) period. Subsequently, pulmonary arterial pressure (Ppa) and the weight ratio of the right ventricle to the left ventricle plus the septum [RV/(LV + S)] were measured, and lungs were obtained for the determination of HIF-1 via Western blot analysis. Both hypoxia and MCT induced temporal increases in the Ppa, the ratio RV/(LV + S) and HIF-1 levels. A close relationship between the Ppa and HIF-1 level was found in both hypoxia- and MCT-treated animals. In addition, the PaO₂ level significantly decreased in rats 1–3 weeks after MCT treatment. These results, along with previous data in the literature, suggest that both chronic hypoxia- and MCT-induced lung hypoxia activate an increase in the production of HIF-1, and result in vascular remodeling and pulmonary hypertension.