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Cancer Research 68, 2781, April 15, 2008. doi: 10.1158/0008-5472.CAN-07-2635
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Manganese Superoxide Dismutase Modulates Hypoxia-Inducible Factor-1{alpha} Induction via Superoxide

Suwimol Kaewpila, Sujatha Venkataraman, Garry R. Buettner and Larry W. Oberley

Free Radical and Radiation Biology Program, Department of Radiation Oncology, Holden Comprehensive Cancer Center, University of Iowa, Iowa City, Iowa

Requests for reprints: Larry W. Oberley, B180 ML, Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA 52242-1181. Phone: 319-335-8015; Fax: 319-335-8039; E-mail: larry-oberley{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays an important role in O2 homeostasis. Numerous observations suggest that changes in reactive oxygen species affect HIF-1{alpha} stabilization and HIF-1{alpha} transcriptional activation in many cell types. The antioxidant enzyme manganese superoxide dismutase (MnSOD) modulates the cellular redox environment by converting superoxide (O2bullet–) to hydrogen peroxide and dioxygen. Previous results from our group have shown that overexpression of MnSOD in MCF-7 cells alters stabilization of HIF-1{alpha} under hypoxic conditions; however, the underlying mechanism(s) is not known. Here, we tested the hypothesis that MnSOD regulates the expression of HIF-1{alpha} by modulating the steady-state level of O2bullet–. We found that decreasing MnSOD with small interfering RNA in MCF-7 cells resulted in (a) an associated increase in the hypoxic accumulation of HIF-1{alpha} immunoreactive protein, (b) a significant increase in the levels of O2bullet– (P < 0.01), but (c) no significant change in the steady-state level of H2O2. Removal of O2bullet– using spin traps ({alpha}-4-pyridyl-1-oxide-N-tert-butylnitrone and 5,5-dimethyl-1-pyrroline N-oxide) or the O2bullet– scavenger Tempol or an SOD mimic (AEOL10113) resulted in a decrease in HIF-1{alpha} protein, consistent with the hypothesis that O2bullet– is an important molecular effector responsible for hypoxic stabilization of HIF-1{alpha}. The evidence from both genetic and pharmaceutical manipulation is consistent with our hypothesis that O2bullet– can contribute to the stabilization of HIF-1{alpha}. [Cancer Res 2008;68(8):2781–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transcription factor hypoxia-inducible factor (HIF) is a key regulator of the cellular response to O2 homeostasis. HIF up-regulates the expression of many genes, including those responsible for angiogenesis, glycolysis, cell growth, cell survival, and metastasis (1, 2). HIF is a heterodimer composed of a constitutively expressed β subunit and an oxygen-regulated {alpha} subunit (3, 4). There are three known forms of HIF: HIF-1, HIF-2, and HIF-3. The immediate response to hypoxia is principally mediated through an increase in the level of HIF-1{alpha}, a ubiquitously expressed protein in most cell types. When O2 is adequate, two prolyl residues at the NH2 terminal activation domain of HIF-1{alpha} are targeted for hydroxylation by appropriate prolyl hydroxylase domain-containing proteins (PHD). Upon hydroxylation, HIF-1{alpha} binds to the Von Hippel-Lindau (pVHL) tumor suppressor protein and leads to its ubiquitination and subsequent degradation via the 26s proteasome (5–7). When there is inadequate O2 in the cell for this hydroxylation reaction, HIF-1{alpha} does not bind to pVHL; thus, it accumulates and translocates to the nucleus where it dimerizes with HIF-1β, leading to formation of the transcription factor HIF. HIF will bind to hypoxia-responsive elements within genes initiating their expression, for example, vascular endothelial growth factor (VEGF) and erythropoietin (8).

Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme (AE) that localizes in the mitochondrial matrix of eukaryotic cells. MnSOD is essential for maintaining normal tissue function. It modulates the intracellular redox environment by dismutating O2bullet– produced by the electron transfer chain in mitochondria forming H2O2 and O2: O2bullet– + O2bullet– + 2H+ -> H2O2 + O2. The majority of tumors have greater steady-state levels of O2bullet– due to loss of MnSOD (9). Multiple studies have shown that reactive oxygen species (ROS) generated from mitochondria can participate in the hypoxia signal transduction pathway that mediates HIF-1{alpha} stabilization (10–12). Lower levels of MnSOD protein and its activity have been found in many types of tumors (13, 14), and one of such sample is MCF-7 cells (15). Moderate overexpression of MnSOD in MCF-7 cells has been shown to suppress hypoxic accumulation of HIF-1{alpha} protein at both 1% and 4% O2 (16). The downstream effects of HIF-1{alpha} suppression by elevated levels of MnSOD activity resulted in a decrease in the secretion of VEGF protein in cells exposed to 1% O2 (16). Alternatively, overexpressing MnSOD or CuZnSOD in A549 human lung epithelial cells does not alter HIF-1{alpha} stabilization under hypoxic conditions, whereas overexpressing GPx1 or catalase decreased HIF-1{alpha} accumulation at low O2 levels (17). In both of these latter studies, changes in the levels of ROS were not reported.

Although the effects of MnSOD on HIF-1{alpha} stabilization have been reported, the mechanism underlying MnSOD-mediated HIF-1{alpha} regulation and the effect of ROS removal on HIF-1{alpha} in response to hypoxia have not been clearly defined. We hypothesize that MnSOD affects the expression of redox-sensitive genes, including HIF-1{alpha}, by modulating ROS levels in cells. We used molecular genetic and chemical approaches for ROS manipulation to analyze the regulation of HIF-1{alpha} during hypoxia. We observed that decreasing the level of MnSOD by small interfering RNA (siRNA) transfection elevated the levels of O2bullet– and induced the accumulation of HIF-1{alpha}. This induction of HIF-1{alpha} was suppressed when O2bullet– was removed using O2bullet– scavengers or an SOD mimic. Here, we propose that MnSOD plays an important role in regulating the accumulation of HIF-1{alpha} during hypoxia by modulating the level of O2bullet–.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and chemicals. AEOL10113 [manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin] was a gift from Dr. James D. Crapo of National Jewish Medical Research Center. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was from Dojindo. Para-hydroxy phenyl acetic acid (pHPA), horseradish peroxidase (HRP), {alpha}-4-pyridyl-1-oxide-N-tert-butylnitrone (POBN), and 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxyl (Tempol) were from Sigma.

Cell culture. Human immortalized nonmalignant mammary epithelial cells, MCF10A, were cultured in DMEM/Ham's F-12 (1:1) supplemented with 5% horse serum, 20 ng/mL epidermal growth factor, 0.01 mg/mL insulin, and 500 ng/mL hydrocortisone. Human breast adenocarcinoma MCF-7 cells were cultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, and 0.1 mmol/L nonessential amino acids. Cells were routinely maintained at 37°C in a humidified atmosphere with 5% CO2.

Induction of hypoxia. Cells were seeded into 60-mm culture dishes; fresh medium was provided before hypoxic or chemical treatments. For hypoxia experiments, the dishes were placed in a hypoxic chamber (Billups-Rothenberg) and flushed with 1% O2 (premixed 1% O2, 5% CO2, 94% N2) for 5 min at 20 L/min, then the gas exchange ports were closed and the chamber was placed in an incubator at 37°C for 4 h.

Inhibition of MnSOD by RNA interference. The predesigned double-stranded siRNA and its complement directed against MnSOD (5'-GGCCUGAUUAUCUAAAAGCTT-3') and the nonspecific siRNA, the commercially available nontargeting siRNA and its complement were purchased from Ambion, Inc. Briefly, 1 x 106 cells were seeded into 60-mm dishes the day before transfection. After 24 h, the media was replaced with OptiMEM (Life Technologies). Cells were then transfected with siRNA using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's instructions. After 24 h, the transfection media was replaced with regular complete media without antibiotics. After 72 h, cells were harvested or treated with hypoxia for further experiments.

Protein harvest for HIF-1{alpha}. Medium was removed from tissue culture dishes, and cells were rinsed twice with cold PBS and then aspirated. Boiling lysis buffer [1% SDS, 1 mmol/L sodium ortho-vanadate, and 10 mmol/L Tris buffer (pH 7.4)] was added to the cells (16). Cells were quickly scraped and transferred into microcentrifuge tubes and boiled for 5 min. The viscosity was reduced by passing the lysates through a 25-gauge needle and then centrifuging at 12,000 x g, 4°C for 10 min, and the supernatants were transferred to a new tube. Protein concentration was determined with Bio-Rad detergent-compatible protein assay.

Western blot analysis. Analysis of HIF-1{alpha} protein used 4% to 20% gradient Tris-HCl polyacrylamide ready-to-use gels (Bio-Rad) and electrotransferred onto a polyvinylidene difluoride membrane. Mouse monoclonal antibody to HIF-1{alpha} (PharMingen/Transduction Laboratories) was used as a primary antibody, whereas mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion) was used as a primary antibody for loading control protein. Goat anti-mouse IgG (PharMingen/Transduction Laboratories) was used as a secondary antibody against both primary antibodies. SDS-polyacrylamide gel (12%) was used for MnSOD Western blot analysis. Equal protein loading was confirmed on immunoblots using rabbit anti-actin antibody (Sigma). Bands were visualized by chemiluminescence (Pierce). All immunoblots were determined from at least three separate experiments. Quantification of band intensity for HIF-1{alpha} was determined using Alpha Imager 2200 program based on integrated density value of each HIF band normalized to GAPDH.

AE activity gels. MnSOD activity was visualized by native PAGE, which is based on the inhibition by SOD of the in-gel reduction of nitroblue tetrazolium (NBT; ref. 18). Briefly, cells were washed with PBS (pH 7.4), and lysates were prepared in NP40 lysis buffer [150 mmol/L NaCl, 1% Nonidet-P40, and 50 mmol/L Tris buffer (pH 8.0)]. Proteins were quantified using the Bio-Rad protein assay. Equal amounts of protein from different samples were loaded onto a polyacrylamide gel (12% running gel with a 5% stacking gel). After electrophoresis, gels were stained with 2.43 mmol/L NBT for 20 min in the dark and then rinsed with distilled water, and 28 µmol/L riboflavin/28 mmol/L TEMED were added and illuminated under fluorescent light. For catalase and glutathione peroxidase GPx activity, 8% and 10% running gel were used. For catalase activity, gels were incubated with 0.003% H2O2 for 10 min and then stained with 2% ferric chloride–2% potassium ferricyanide solution (19). For GPx activity, gels were soaked in 1 mmol/L reduced glutathione for 30 min, incubated with 0.008% cumene hydroperoxide for 10 min, and then finally stained with 1% ferric chloride–1% potassium ferricyanide solution (19).

AE activity assays. SOD activity was also measured by the modified NBT method, as described previously (20). Briefly, SOD activity was determined spectrophotometrically at 560 nm by measuring the reduction of NBT. The O2bullet– generated from the xanthine and xanthine oxidase system reduces NBT. The reduction of NBT is competitively inhibited in the presence of SOD. The amount of protein that inhibits the reduction of NBT to 50% of maximum is defined as one unit of SOD activity. MnSOD activity was determined in the presence of 5 mmol/L sodium cyanide. CuZnSOD activity was calculated by subtracting MnSOD activity from total SOD activity.

Superoxide radical anion formation in cultured cells. Electron paramagnetic resonance (EPR) spin trapping with DMPO was used to detect O2bullet–. This technique involves an addition reaction of a short-lived radical to a diamagnetic compound (spin trap) to form a more stable free radical product (spin adduct), which can be studied by EPR. The intensity of the spin adduct signal corresponds to the amount of short-lived radicals trapped; the hyperfine couplings of the spin adduct are characteristic of the original trapped radical. In brief, cells were washed with PBS and incubated with 100 mmol/L DMPO in chelated PBS (pH 7.4; ref. 21) for 15 min. The cells were then transferred to a TM quartz flat cell, and EPR spectra were recorded using a Bruker EMX spectrometer equipped with a TM cavity. EPR spectra were obtained as an average of 15 scans with a modulation amplitude of 1 G, scan rate of 80 G/81 s, receiver gain of 104 to 106, microwave power of 40 mW, and modulation frequency of 100 kHz. The EPR peak heights are in arbitrary units.

Hydrogen peroxide determination. Extracellular H2O2 released from MCF-7 cells was measured by a fluorometric assay using pHPA in the presence of HRP, as previously described (22). Briefly, this method used the fact that H2O2 reacts with HRP-forming compound I, which in turn reacts with pHPA, forming a stable fluorescent dimer, [pHPA]2. Cell medium was removed, and the cell monolayer was washed thrice with HBSS buffer. The medium was then replaced with phenol red-free HBSS (1.0 mL) supplemented with 6.5 mmol/L glucose, 1 mmol/L HEPES, 6 mmol/L sodium bicarbonate, 1.6 mmol/L pHPA, and 95 µg/mL HRP. The H2O2 was allowed to accumulate in the modified HBSS for 1 h. The released H2O2 was followed spectrofluorometrically by measuring the dimer formed at excitation and emission wavelengths of 323 and 400 nm, respectively. The fluorescence intensity of each sample was corrected for any changes in pH and compared with standard concentrations of H2O2 determined by absorbance at 240 nm.

Clonogenic survival. Cells were washed, trypsinized, and plated immediately after hypoxia treatment with and without chemical agent treatments into 60-mm dishes. The dishes were maintained in an incubator at 21% O2 for 14 d to allow colony formation. The colonies were fixed with 70% ethanol for 5 min and stained with Coomassie blue for 5 min. Those colonies containing >50 cells were scored. Cell survival fraction (SF) was calculated as follows: SF = colonies formed / (cells seeded x PE), where PE is plating efficiency, i.e., (number of colonies formed) / (number of cells seeded) x 100.

Statistical analysis. Data are mean ± SEM from three independent experiments. Statistical analyses to determine the differences between means were performed using one-way ANOVA, followed by a post-hoc Tukey test or Student's t test. P < 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MnSOD protein expression was suppressed by transient siRNA transfection. To determine whether MnSOD could affect the expression of HIF-1{alpha} under hypoxic conditions, we first manipulated MnSOD levels in human breast adenocarcinoma MCF-7 cells using specific RNA interference. The protein level of MnSOD in MCF-7 cells was observed to be lower than those of immortalized, nonmalignant MCF10A breast cells (Fig. 1A ). In MCF-7 cells, MnSOD protein expression was found to be suppressed by siRNA in a time-dependent and concentration-dependent manner (Fig. 1B and C). siRNA against MnSOD showed suppression of protein within 24 hours after transfection with maximal decrease at 72 hours. Nontargeting siRNA transfected cells were similar to untransfected control (Fig. 1B and C, Neg). The transfection conditions of 300 pmol for 72 hours were selected for use in all subsequent experiments. The activity of MnSOD in cells transfected with siRNA was below the limit of detection of the spectroscopic-based assay. However, the suppression of the activity of MnSOD could be shown by nondissociating native gel electrophoresis (Fig. 1D). No changes in the activities of other AEs, such as CuZnSOD, catalase, or GPx, as measured by activity gels, were observed (data not shown).


Figure 1
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  		Figure 1. Transient siRNA knockdown of MnSOD. A, Western blot analysis of whole-cell lysates showing MnSOD protein levels of immortalized nonmalignant breast cells MCF-10A and human breast adenocarcinoma MCF-7. B and C, MCF-7 cells were transfected with MnSOD siRNA and nontargeting siRNA (siNeg) at different times and concentrations. Whole-cell lysates were analyzed by Western blot for MnSOD expression using actin as a protein loading control. MnSOD protein expression was compared relative to untransfected (control) or nontargeting siRNA. D, MnSOD activities were determined in MCF-7 cells transfected with 300 pmol siRNA for 72 h by nondissociating electrophoresis (12% gels) stained for SOD activity. All results are representative of at least three separate experiments.

 
Inhibition of MnSOD by siRNA increased O2bullet– levels and induced HIF-1{alpha} accumulation in cells exposed to 1% O2. We next determined the level of HIF-1{alpha} protein induction in MCF-7 cells after being transiently transfected with siRNA against MnSOD, nontargeting siRNA, or exposure to the transfection agent alone. The transfected cells were exposed to 1% O2 for 4 h, and the relative levels of MnSOD protein were determined. After treatment with MnSOD siRNA relative to nontargeting siRNA or transfection reagent control, a decrease in MnSOD protein and a modest increase of HIF-1{alpha} protein was observed (Fig. 2A ). This was further confirmed by the quantitation of the blot intensities. These results suggest that changes in MnSOD levels had an effect on HIF-1{alpha} accumulation under hypoxic conditions in MCF-7 cells.


Figure 2
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  		Figure 2. Inhibition of MnSOD in MCF-7 cells by siRNA significantly increased O2bullet– levels and an induction of HIF–1{alpha} protein under 1% O2 conditions. A, cells were transfected with siRNA against MnSOD or nontargeting siRNA as a nontargeting control (300 pmol, 72 h) or exposed to transfection reagent alone (control). Cells were exposed to hypoxia (1% O2) for 4 h. Whole-cell lysates were analyzed for the expression of MnSOD and HIF-1{alpha} protein by Western blot analysis. Relative band intensities for HIF-1{alpha} are presented under the blots. B, representative EPR spectra, measured at 21% and 1% O2, showing the DMPO-OH spin adduct (aN = aH = 14.9 G) normalized to the protein. The intensity of the DMPO-OH signal corresponds to the relative rate of O2bullet– formation. C, the EPR peak height (normalized to the amount of protein) measured from cells transfected with siRNA against MnSOD is significantly different from nontargeting siRNA and control (i.e., untransfected cells; P < 0.01). D, extracellular H2O2 accumulation was determined by pHPA fluorescence assay (P > 0.05 relative to nontargeting siRNA). All results are representative of at least three separate experiments.

 
To evaluate the functional consequences of MnSOD-mediated O2bullet– induction of HIF-1{alpha}, we determined whether inhibiting MnSOD actually results in an increased level of O2bullet–, the substrate of MnSOD. MCF-7 cells were transfected with siRNA followed by exposure to 1% O2. Free radical production was assessed by EPR by measuring the intensities of the DMPO-OH spin adduct, which corresponds to the relative rates of O2bullet– formation, which is inhibitable by SOD (not shown). After 4 hours of hypoxia, cells accumulated EPR-detectable DMPO-OH adducts (Fig. 2B). EPR spectra from cells transfected with MnSOD siRNA showed a greater peak height of the DMPO-OH spin adduct relative to nontargeting siRNA and untransfected controls. There were no significant differences in the intensities of the spectra observed from nontargeting siRNA and untransfected control. Quantitation of the spectral peak heights normalized to the amount of protein indicated a significant increase in accumulation of DMPO-OH measured from cells transfected with MnSOD siRNA relative to nontargeting siRNA (P < 0.01; Fig. 2C). Extracellular accumulation of H2O2 from MnSOD siRNA transfected cells showed no significant difference relative to nontargeting siRNA transfected cells (Fig. 2D). These results show that the suppression of MnSOD by siRNA significantly increased the level of O2bullet– and concomitantly increased HIF-1{alpha} induction under hypoxia in MCF-7 cells. The increase in accumulation of HIF-1{alpha} protein when MnSOD is decreased in this cell line suggests that O2bullet– plays a role in the regulation of HIF-1{alpha}.

Scavenging of O2bullet– by the spin traps POBN and DMPO suppressed HIF-1{alpha} under 1% O2 conditions. By using siRNA, we showed that a decreased level of MnSOD resulted in a significant increase in the steady-state level of O2bullet– and an induction of HIF–1{alpha} protein. To determine whether O2bullet– is an important molecular species responsible for the induction of HIF-1{alpha} during hypoxia, levels of O2bullet– were lowered in MCF-7 cells with the spin trapping agents POBN or DMPO. Different concentrations of POBN or DMPO (33–100 mmol/L) were introduced to the cells under both 21% and 1% O2 conditions, and HIF-1{alpha} protein was determined. These high concentrations are necessary because of their low rate of reaction with O2bullet– compared with the naturally occurring SODs. POBN was present through the 4-hour incubation. However, because of its high reactivity and propensity to form oxidation products, DMPO was added only for the final hour of the hypoxic incubation. As expected, HIF-1{alpha} protein was detectable only under 1% O2. Cells treated with these spin traps had decreased levels of HIF-1{alpha} after the hypoxic incubation. HIF-1{alpha} protein induction seemed to be decreased in a spin trap concentration-dependent manner (Fig. 3A ).


Figure 3
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  		Figure 3. Scavenging of O2bullet– by spin traps POBN or DMPO suppressed HIF-1{alpha} protein under 1% O2 conditions. A, MCF-7 cells were incubated in the absence (0 mmol/L) and presence (33–100 mmol/L) of POBN under 21% or 1% O2 for 4 h. For the DMPO experiments, the spin trap was added after cells were pretreated with 1% O2 for 3 h; then the incubation was continued for 1 h under 1% O2. Whole-cell lysates were analyzed for expression of HIF-1{alpha} protein by Western blot analysis. B, the EPR peak height of the DMPO-OH spin adduct normalized to the amount of protein was significantly decreased from cells treated with DMPO (100 mmol/L at both 21% and 1% O2) during the hypoxic incubation relative to untreated control. For these experiments, the media with spin trap for the hypoxic incubation was removed from the cells and replaced with chelated PBS containing fresh DMPO (100 mmol/L). Spectra were collected as in Materials and Methods.

 
To monitor other consequences of introducing spin trapping agents during the hypoxic incubation, the ability of cells to produce ROS after the hypoxic incubation was examined. Relative levels of ROS were assessed under both normoxia and hypoxia. Media was removed, and fresh DMPO (100 mmol/L) in chelated-PBS was introduced. The signal height from the EPR spectra normalized to the amount of protein was lower from those cells exposed to spin trap (100 mmol/L) during the hypoxic incubation (Fig. 3B). These results are consistent with O2bullet– being involved in HIF-1{alpha} induction under hypoxia.

Scavenging of O2bullet– by Tempol affected HIF-1{alpha} protein induction after exposure to 1% O2. Data from the spin trapping experiments suggested a role for O2bullet– in the regulation of the hypoxic accumulation of HIF-1{alpha}. However, spin traps do not mimic SOD activity because they do not produce H2O2 as a product upon their reaction with O2bullet–. In another approach to test our hypothesis, we used Tempol as an O2bullet– scavenger. Tempol is a stable nitroxide radical that has SOD mimetic activity (23); it dismutes two O2bullet– radicals, producing H2O2 and O2. When concentration is sufficiently high, it has been reported that Tempol will react with the protonated form of O2bullet– (hydroperoxyl radical, bulletOOH) to produce H2O2 and oxoammonium salts (24). Cells were treated with various concentrations of Tempol (0.1–40 mmol/L) under hypoxic conditions. Tempol induced HIF-1{alpha} accumulation under hypoxic conditions as the concentration increased (Fig. 4A ). This effect seemed to be dose-dependent because, at 10 mmol/L Tempol, a decrease in HIF-1{alpha} protein was observed; a further decrease was seen with 20 mmol/L, and HIF-1{alpha} was undetectable at 40 mmol/L. The level of MnSOD protein was not affected. After hypoxia, the ability of cells to generate DMPO-OH signal was determined. Cells treated with 0.1 or 1 mmol/L Tempol generated significantly less DMPO-OH relative to untreated controls at 1% O2 (P < 0.05). When higher concentrations of Tempol (10, 20, and 40 mmol/L) were used, the signal for DMPO-OH was not visible because the peaks were masked by the Tempol signal (Fig. 4B). These results suggest that the level of O2bullet– generated by cells treated with Tempol was significantly decreased relative to untreated cells at 1% O2. Extracellular levels of H2O2 were not altered at lower concentrations of Tempol (0.1–10 mmol/L), but increased with 20 and 40 mmol/L, relative to untreated cells at 1% O2 (P < 0.05; Fig. 4C). These observations are consistent with O2bullet– being an important molecular effector underlying hypoxic HIF-1{alpha} stabilization.


Figure 4
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  		Figure 4. Scavenging of O2bullet– by Tempol affected HIF-1{alpha} protein accumulation under 1% O2 conditions. A, MCF-7 cells were treated with Tempol at different concentrations (0.1–40 mmol/L) at 1% O2 for 4 h. Whole-cell lysates were analyzed for the expression of HIF-1{alpha} and MnSOD protein by Western blot analysis. B, EPR signal height of DMPO-OH normalized to the amount of protein obtained from cells treated with various concentration of Tempol (P < 0.05 relative to 1% O2 control). #, DMPO-OH adduct peaks in the presence of 10 to 40 mmol/L Tempol were masked by the Tempol EPR signal and thus could not be quantified. C, extracellular H2O2 formation was determined in Tempol-treated cells by the pHPA fluorescence assay (P < 0.05 relative to 1% control).

 
SOD mimic suppressed HIF-1{alpha} induction under 1% O2. To further analyze the effects of ROS removal and/or ROS generation in MCF-7 cells, we used the AEOL10113, a small molecular weight manganese-containing porphyrin that has potent SOD mimic activity (25). It has been reported that AEOL10113 can reduce hypoxia-induced O2bullet– levels and VEGF production by macrophages (26). Different concentrations of the compound were introduced to MCF-7 cells at 21% O2 for 4 h, then cells were treated with hypoxia, and HIF-1{alpha} protein was determined. At a low concentration of AEOL10113 (10 µmol/L), HIF-{alpha} was found to be decreased; an additional decrease was seen with 20 µmol/L mimetic. However, when AEOL10113 concentrations were increased further, HIF-1{alpha} levels increased (Fig. 5A ). The changes in the fold intensities were measured by integrated density values. Consistent with its SOD mimetic activity, the level of O2bullet– decreased (Fig. 5B) and the level of H2O2 increased (Fig. 5C; ref. 27). Interestingly, at 50 µmol/L AEOL10113, a concentration wherein O2bullet– was lowest relative to untreated cells (P < 0.05), the accumulation of H2O2 was greatest. This biphasic response parallels to that observed by Wang et al. (16).


Figure 5
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  		Figure 5. SOD mimic suppressed HIF-1{alpha} expression under 1% O2 conditions. A, different concentrations (10–200 µmol/L) of AEOL10113 were added to MCF-7 cells at 21% O2 for 4 h followed by incubation at 1% O2 for 4 h. HIF-1{alpha} protein expression was analyzed from whole-cell lysates by Western blot. Relative band intensities for HIF-1{alpha} are presented under the blots. B, quantified data from the EPR spectra normalized to the amount of protein obtained from cells treated with different concentrations of AEOL10113 (P < 0.05 relative to 1% control). C, extracellular H2O2 accumulation was determined by the pHPA fluorescence assay (P < 0.05 relative to 1% untreated control).

 
Changes in superoxide levels affected clonogenic survival of MCF-7 cells. In spin trapping and O2bullet– scavenger experiments, we observed a significant decrease in DMPO-OH signals, which implies that O2bullet– levels in the cells had been altered. We hypothesized that the changes in O2bullet– levels induced by these spin traps and O2bullet– scavengers may be harmful and thus contribute to cell mortality. To test the hypothesis, we evaluated the cytotoxicity of these agents by determining clonogenic survival of the cells after treatment during the exposure to hypoxia. Cells treated with either POBN or DMPO during 1% O2 exposure showed no significant difference in survival fractions relative to untreated control cells (P > 0.05), but the surviving fractions were significantly decreased relative to untreated cells at 21% O2 (P < 0.01; Fig. 6A and B ). Cells treated with either 1% O2 or 1% O2 + Tempol showed a significant decrease in survival fraction (P < 0.01) relative to untreated cells at 21% O2; cells treated with 1% O2 + Tempol exposure showed a significant decrease in survival fraction relative to control 1% (P < 0.01) only when the concentrations of Tempol were higher than 10 mmol/L (Fig. 6C). However, cells treated with AEOL10113 at 1% O2 conditions showed no significant difference in survival fraction compared with untreated cells at 21% and 1% O2; only cells treated with 1% O2 + AEOL10113 at 200 µmol/L exposure showed a significant decrease in survival fraction relative to 1% O2 + AEOL10113 at 20 µmol/L (P < 0.05; Fig. 6D).


Figure 6
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  		Figure 6. Clonogenic survival after treatment with superoxide removal by spin traps, O2bullet– scavenger, and SOD mimic. A, MCF-7 cells were treated with 100 mmol/L POBN under both 21% and 1% O2 for 4 h. B, DMPO (100 mmol/L) was added after cells were pretreated with 1% O2 for 3 h, and then the incubation was continued for 1 h under 1% O2; P < 0.01 compared with 21% control and P > 0.05 compared with 1% control. C, cells were treated with different concentrations of Tempol at 1% O2; P < 0.01 compared with 21% and 1% O2 control respectively. D, cells were pretreated with AEOL10113 at 21% O2 for 4 h, and then 1% O2 incubation was continued for 4 h; P > 0.05 compared with 21% and 1% O2 control and P < 0.05 compared with 1% O2 + 20 µmol/L AEOL10113. To examine the effect of O2bullet– removal under hypoxia on cell proliferation, clonogenic assays were performed, as in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years, much effort has been devoted to the use of respiratory inhibitors or {rho}0 cells to examine the role of the mitochondrial electron transport chain in the regulation of HIF-1{alpha}. It is likely that mechanisms of O2 sensing and signaling during hypoxia are associated with mitochondrial ROS generation and involve different pathways in different cell types (10, 17, 28). Work using genetic approaches has shown that HEK293 cells transfected with siRNA against the Rieske iron-sulfur protein of mitochondria complex III failed to stabilize HIF-1{alpha} protein during hypoxia. In addition, both wild-type human fibroblasts and cells that had an impairment of oxidative phosphorylation exhibited an increase in HIF-1{alpha} protein stabilization when exposed to hypoxia (1.5% O2), which was prevented by the addition of myxothiazol. The authors concluded that oxidative phosphorylation is not required for the hypoxic stabilization of HIF-1{alpha} but mitochondrial ROS are needed (17). Therefore, ROS play a major role in stabilizing HIF-1{alpha}.

The exact molecular nature of ROS responsible for regulation of HIF-1{alpha} under hypoxia is not clear. Wang et al. found that overexpression of MnSOD resulted in a biphasic effect on HIF-1{alpha} protein levels (16). They showed that with relatively low overexpression of MnSOD, HIF-1{alpha} decreased. Because an increase in MnSOD would lower the steady-state level of superoxide, this observation suggests that superoxide may play a role in the stabilization of HIF-1{alpha} protein. However, when MnSOD was highly overexpressed, HIF-1{alpha} was again present. Because high levels of MnSOD can lead to greater fluxes of H2O2, this suggests that H2O2 may also regulate HIF-1{alpha}. Goyal et al. found that overexpression of a NADPH oxidase 1 (Nox1), which generates high fluxes of O2bullet–, in human lung adenocarcinoma A549 cells resulted in accumulation of HIF-1{alpha} in normoxia (29); under hypoxia (1% O2), an additional increase was observed. These effects could be reversed by the flavoprotein inhibitor diphenylene iodonium or by catalase. These observations are consistent with high levels of H2O2 being able to activate HIF-1{alpha}. Therefore, the appearance of HIF-1{alpha} at higher levels of MnSOD, as well as with the activation of Nox1, suggests that high levels of H2O2 can lead to accumulation of HIF-1{alpha}. To address these possible roles of ROS in stabilizing HIF-1{alpha}, we carried out experiments using ROS scavengers.

When MCF-7 cells were exposed to nontoxic concentrations of Tempol (<10 mmol/L), the protein level of HIF-1{alpha} increased (Fig. 4A). Concomitantly, the level of O2bullet–, as seen by the intensity of the DMPO-OH signal, was decreased (Fig. 4B). At nontoxic concentrations of Tempol, there was no change in the level of H2O2 (Fig. 4C); concentrations of Tempol (>1 mmol/L) are toxic, as seen by decreased cell survival (Fig. 6C). Tempol is a redox active compound and may well intercept the ferryl state of PHD, not allowing the hydroxylation of HIF-1{alpha} to occur. That Tempol allows HIF-1{alpha} to accumulate may explain the many positive in vivo observations reported with this compound (30). Thus, firm conclusions on the identity of a specific ROS that regulates HIF-1{alpha} cannot be made from these observations. To better probe for the identity of ROS, we carried out experiments with an SOD mimic, AEOL10113 (24). This SOD mimic had no effect on cell survival (Fig. 6D).

To address better the role of ROS in the modulation of HIF-1{alpha}, we introduced varying levels of AEOL10113 to cells in combination with exposure to hypoxia. Similar to the observations of Wang et al. (16), we observed a biphasic effect in the accumulation of HIF-1{alpha} with varying concentration of AEOL10113. At low concentrations of SOD mimic, we observed decreased levels of HIF-1{alpha} in MCF-7 cells and a concomitant decrease in the levels of O2bullet– during hypoxia as studied by EPR spin trapping (Fig. 5A and B). Whereas at higher concentrations of SOD mimic, corresponding increases in H2O2 were seen with parallel increases in HIF-1{alpha} protein. Because the SOD mimic altered the levels of both O2bullet– and H2O2, the precise roles of O2bullet– and H2O2 cannot be deconvoluted. Therefore, we took another approach to alter the endogenous levels of O2bullet– and MnSOD.

In the experiments with Tempol and AEOL10113, the goal was to increase the effective SOD-like activity in cells. To specifically decrease the endogenous MnSOD activity, we used siRNA against MnSOD. This should result in an increase in the steady-state level of O2bullet–, which should lead to an increase in HIF-1{alpha}. Indeed, upon introduction of siRNA, we observed the anticipated lowering of MnSOD and an increase in both O2bullet– and HIF-1{alpha} (Fig. 2A and C). There was no detectable change in the level of H2O2 (Fig. 2D). These observations point directly to O2bullet– as a modulator of HIF-1{alpha} stabilization during hypoxia.

To provide additional evidence for the role of O2bullet– in modulating HIF-1{alpha}, we used the spin traps POBN and DMPO as scavengers of O2bullet–. Lowering the steady-state level of O2bullet– should lower HIF-1{alpha} under hypoxia. We found that both POBN and DMPO lowered the level of HIF-1{alpha} protein (Fig. 3A). Neither POBN nor DMPO were significantly toxic to the cells under our experimental conditions (Fig. 6A and B). Taking all observations together, it is clear that O2bullet– has a major role in regulating HIF-1{alpha}.

Here, we propose that MnSOD plays an important role in regulating HIF-1{alpha} accumulation during hypoxia by modulating the levels of O2bullet–. To our knowledge, this is the first demonstration that MnSOD regulates HIF-1{alpha} via O2bullet–. These results should provide a better understanding of the biological role of MnSOD in regulating HIF-1{alpha} in tumor cells.


   Acknowledgments
 
Grant support: Milheim Foundation 2005-16. S. Kaewpila was partially supported by Ministry of Science under Royal Thai Government and Free Radical and Radiation Biology Program of University of Iowa. NIH CA66081 and CA132850.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors thank Dr. James D. Crapo, Department of Medicine, National Jewish Medical and Research Center, who provided the SOD mimic; Dr. Rebecca E. Oberley for her help; Dr. Melissa L.T. Teoh for technical help with siRNA experiments; Dr. Douglas R. Spitz of the Antioxidant Enzyme Core for technical advice on MnSOD activity assays; and Dr. Terry D. Oberley, University of Wisconsin School of Medicine and Public Health, for his help in preparing the manuscript.

Received 7/11/07. Revised 1/14/08. Accepted 2/19/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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