Limits...
Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis.

Bergström AL, Fog K, Sager TN, Bruun AT, Thirstrup K - ISRN Neurosci (2013)

Bottom Line: In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12).Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress.In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegeneration, Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.

ABSTRACT
The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1 α to the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

No MeSH data available.


Related in: MedlinePlus

Cellomics images showing the algorithm used for quantification of nuclear and cytoplasmic levels of HIF-1α. Raw images without the applied algorithm used to define cells are shown in the top panel and images showing the algorithm are shown in the lower panel. Circ is defined by the outline of the Hoechst-staining and thus represents the nuclear region. Ring is defined as a certain radius surrounding the Circ region and thus represents the cytoplasmic region of the cell. The Hoechst fluorescence and the HIF-1α fluorescence are recorded in two different channels.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4061615&req=5

fig1: Cellomics images showing the algorithm used for quantification of nuclear and cytoplasmic levels of HIF-1α. Raw images without the applied algorithm used to define cells are shown in the top panel and images showing the algorithm are shown in the lower panel. Circ is defined by the outline of the Hoechst-staining and thus represents the nuclear region. Ring is defined as a certain radius surrounding the Circ region and thus represents the cytoplasmic region of the cell. The Hoechst fluorescence and the HIF-1α fluorescence are recorded in two different channels.

Mentions: In order to quantify the level of HIF-1-α in the nucleus and cytoplasm, respectively, the Compartmental Analysis BioApplication for Cellomics ArrayScan was used. Cells were grown in 96-well plates, treated with compounds for the indicated times, and fixed with 4% paraformaldehyde. HIF-1-α was detected by immunocytochemistry as described above. An algorithm was set up and the level of HIF-1α-Cy3-fluorescence in the nuclear region (Circ, see Figure 1(b)) and the cytosolic region (Ring, defined as a fixed diameter region surrounding the nucleus, see Figure 1(b)) was quantified. The ratio of nuclear to cytosolic HIF-1α is here representing the level of nuclear translocation of HIF-1α when compared to untreated control.


Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis.

Bergström AL, Fog K, Sager TN, Bruun AT, Thirstrup K - ISRN Neurosci (2013)

Cellomics images showing the algorithm used for quantification of nuclear and cytoplasmic levels of HIF-1α. Raw images without the applied algorithm used to define cells are shown in the top panel and images showing the algorithm are shown in the lower panel. Circ is defined by the outline of the Hoechst-staining and thus represents the nuclear region. Ring is defined as a certain radius surrounding the Circ region and thus represents the cytoplasmic region of the cell. The Hoechst fluorescence and the HIF-1α fluorescence are recorded in two different channels.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4061615&req=5

fig1: Cellomics images showing the algorithm used for quantification of nuclear and cytoplasmic levels of HIF-1α. Raw images without the applied algorithm used to define cells are shown in the top panel and images showing the algorithm are shown in the lower panel. Circ is defined by the outline of the Hoechst-staining and thus represents the nuclear region. Ring is defined as a certain radius surrounding the Circ region and thus represents the cytoplasmic region of the cell. The Hoechst fluorescence and the HIF-1α fluorescence are recorded in two different channels.
Mentions: In order to quantify the level of HIF-1-α in the nucleus and cytoplasm, respectively, the Compartmental Analysis BioApplication for Cellomics ArrayScan was used. Cells were grown in 96-well plates, treated with compounds for the indicated times, and fixed with 4% paraformaldehyde. HIF-1-α was detected by immunocytochemistry as described above. An algorithm was set up and the level of HIF-1α-Cy3-fluorescence in the nuclear region (Circ, see Figure 1(b)) and the cytosolic region (Ring, defined as a fixed diameter region surrounding the nucleus, see Figure 1(b)) was quantified. The ratio of nuclear to cytosolic HIF-1α is here representing the level of nuclear translocation of HIF-1α when compared to untreated control.

Bottom Line: In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12).Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress.In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegeneration, Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.

ABSTRACT
The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1 α to the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

No MeSH data available.


Related in: MedlinePlus