Notes
The 5-Lipoxygenase Inhibitor Zileuton Confers Neuroprotection against Glutamate Oxidative Damage by Inhibiting Ferroptosis
2015 Volume 38 Issue 8 Pages 1234-1239
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2015 Volume 38 Issue 8 Pages 1234-1239
5-Lipoxygenase (5-LOX) inhibitors have been shown to be protective in several neurodegenerative disease models; however, the underlying mechanisms remain unclear. We investigated whether 5-LOX inhibitor zileuton conferred direct neuroprotection against glutamate oxidative toxicity by inhibiting ferroptosis, a newly identified iron-dependent programmed cell death. Treatment of HT22 mouse neuronal cell line with glutamate resulted in significant cell death, which was inhibited by zileuton in a dose-dependent manner. Consistently, zileuton decreased glutamate-induced production of reactive oxygen species but did not restore glutamate-induced depletion of glutathione. Moreover, the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-fmk) neither prevented HT22 cell death induced by glutamate nor affected zileuton protection against glutamate oxidative toxicity, suggesting that zileuton did not confer neuroprotection by inhibiting caspase-dependent apoptosis. Interestingly, glutamate-induced HT22 cell death was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. Moreover, zileuton protected HT22 neuronal cells from erastin-induced ferroptosis. However, we did not observe synergic protective effects of zileuton and ferrostatin-1 on glutamate-induced cell death. These results suggested that both the 5-LOX inhibitor zileuton and the ferropotosis inhibitor ferrostatin-1 acted through the same cascade to protect against glutamate oxidative toxicity. In conclusion, our results suggested that zileuton protected neurons from glutamate-induced oxidative stress at least in part by inhibiting ferroptosis.
Glutamate, an important excitatory transmitter in the central nervous system, essentially contributed to the pathogenesis of a variety of neurological diseases. When excessively released, glutamate induces both receptor-dependent excitotoxicity and non-receptor-mediated oxidative toxicity, which are implicated in a number of neurodegeneration diseases, such as stroke.1–6) Thus, targeting glutamate-induced oxidative toxicity is a promising strategy for treating neurological diseases.1,2)
Lipoxygenases (LOXs) are the enzymes responsible for lipid peroxidation. In the glutamate-induced neurotoxic model, glutamate-induced depletion of glutathione (GSH) leads to lipid peroxidation via the activation of 12,15-LOX, which in turn triggers downstream death signals in neurons.1) Emerging data suggest that LOXs play essential roles in the pathogenesis of various neurological diseases.7–11) For instance, the expression and activity of 12,15-LOX are upregulated in the brain following cerebral ischemia.7) Consequently, 12,15-LOX inhibitors ameliorate brain infarct damage via direct neuroprotection against oxidative stress.7,8) 5-LOX is an important lipoxygenase, whose expression and activity are also elevated in the brain following cerebral ischemia.8,11) Previous studies have shown that 5-LOX inhibition protects against cerebral ischemia by attenuating post-ischemic neuroinflammation.9) Notably, 5-LOX induction is majorly located in dying neurons in the ischemic brain, suggesting that 5-LOX may contribute to neuronal death induced by oxidative stress.10,11) Surprisingly, it has not been investigated if 5-LOX inhibitors confer direct neuroprotection against oxidative stress.
HT22 is a mouse hippocampal neuronal cell line. Glutamate-induced neurotoxic in HT22 has been widely used as an in vitro model to study oxidative stress-induced neurotoxicity associated with both acute and chronic insults.12) Since HT22 cells lack functional glutamate receptors, neurotoxicity induced by glutamate in HT22 cells is exclusively attributed to oxidative toxicity. Zileuton, the only approved 5-LOX inhibitor as a treatment for asthma, is wildly utilized as a selective tool to evaluate the role of 5-LOX.13) By using the model of oxidative glutamate toxicity in HT22 cells, we investigated if zileuton conferred direct neuroprotection against oxidative toxicity.
Ferroptosis is a newly identified iron-dependent cell death pathway, which is initially identified to be induced in tumor cell lines, including some cell lines overexpressing RAS, by a number of ferroptosis-inducing agents. Emerging evidence indicates that ferroptosis is a mechanism underlying oxidative glutamate toxicity in brains and neurons.14,15) Indeed, excessive glutamate depletes cellular GSH in HT22 cells,16,17) which is exactly the characteristic of ferroptosis induced by class I ferroptosis-inducing agents.18) Since ferroptosis is characterized by the accumulation of lethal levels of lipid peroxidation,19) we investigated if 5-LOX inhibition protected neurons against glutamate oxidative stress by inhibiting ferroptosis.
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL Co. (Grand Island, NY, U.S.A.). Zileuton, dimethyl sulfoxide (DMSO), glutamate, erastin and ferrostatin-1 were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-fmk) was obtained from BD Biosciences Pharmingen (CA, U.S.A.). Reactive Oxygen Species Assay Kit and Total Glutathione Assay Kit were purchased from Beyotime Company (Jiangsu, China).
Glutamate-Induced Oxidative Toxicity and Cell Viability Assay MouseHippocampal HT22 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 µg/mL penicillin–streptomycin and incubated in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. To induce oxidative toxicity, cells were grown on 24-well plate for 24 h and then treated with glutamate (5 mM) in the presence or absence of different concentrations of zileuton (1, 10, 50, and 100 µM), the caspase inhibitor ZVAD-fmk (10 µM) or the ferroptosis inhibitor ferrostatin-1 (12.5 µM). The doses for ZVAD-fmk and ferrostatin-1 were selected based on previous publications.15,20) Cell viability were determined at 24 h after treatment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, MTT was added into culture media at a final concentration of 500 nM and cells were kept in the CO2 incubator for 2 h. Then, cells were lysed with DMSO to dissolve formazan converted from MTT by mitochondria. The absorbance measured photometrically at 570 nm was positively correlated with cell viability. Final results were expressed as percentages of viability of control cells without glutamate treatment.
Reactive Oxygen Species (ROS) MeasurementHT22 cells were grown on 24-well plates for 24 h at 37°C. Cells were treated with 5 mM glutamate and/or 10 µM zileuton for 12 h. Then, cells were incubated with serum-free DMEM containing 5 µM 5- (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (H2DCFDA-DA) in a CO2 incubator. At 30 min after incubation, H2DCFDA-DA reacted with ROS to form a fluorescent compound and the medium was replaced with serum-free DMEM without H2DCFDA-DA. Micrographs were taken under a fluorescent microscope.
GSH DeterminationAfter grown on 6-well plates for 24 h at 37°C, HT22 cells were treated with 5 mM glutamate or erastin (500 nM) in the presence or absence of zileuton at 10 or 50 µM for 12 h. Cells were rinsed with phosphate-buffered saline for three times, lysed with radio immunoprecipitation assay (RIPA) buffer and kept on ice for 30 min. After centrifugation at 13200 rpm/min for 20 min, supernatants were collected. Protein concentrations were determined with a commercial BCA kit (Pierce, Thermo Fisher Scientific, Rockford, IL, U.S.A.). Total GSH was assayed using a commercial Kit per the manual provided by the manufacturer (Beyotime Institute of Biotechnology, Nantong, China). Briefly, 10 µL supernatants or GSH standard were added to 150 µL reaction solution containing 0.044 mg/mL 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and glutathione reductase. Fifty microliters reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.16 mg/mL) was added after reactions lasted for 5 min. The absorbance was monitored photometrically at 412 nm with Microplate Reader (Infinite M200 PRO, Tecan, Switzerland) at 5-min intervals. GSH concentrations were calculated from the GSH standard curves. After normalizing protein concentrations, final results were presented as percentages of GSH contents in control cells without glutamate treatments.
Ferroptosis Induction and Cell Viability AssayHT22 cells were grown on 24-well plates at 37°C for 24 h. Then, cells were treated with the ferroptosis inducer erastin (500 nM) in the presence or absence of different concentrations of zileuton (1–100 µM). The doses for erastin was selected based on the previous publication.15) At 24 h after treatment, MTT assay was performed to determine cell viability as described earlier. Final results were expressed as percentages of viability of control cells without erastin treatment.
Free Radical Scavenging Activity AssayFree radical scavenging activity was assayed as previously reported with slight modification.19) Zileuton dissolved in DMSO (10 µL) was added to 390 µL of the stable radical 2,2-diphenyl-1-picrylhydrazyl solution (DPPH, 25 mg/mL in menthol). The mixtures were inverted several times, kept for 10 min and then aliquoted to 96-well plates. Absorbance at 517 nm was recorded with a microplate reader (Infinite M200 PRO, Tecan). The final results were expressed as the ratios to absorbance of control DPPH solution with addition of vehicle (Con).
Statistical AnalysisThe numerical data were presented as mean±standard error of the mean (S.E.M.) and analyzed with SPSS 17.0. The significance of differences between experimental groups was determined with one-way ANOVA followed by Turkey’s post-hoc test. A value of p<0.05 was considered statistically significant and p<0.01 as highly significant.
To investigate the direct neuroprotection conferred by zileuton against oxidative stress, HT22 neuronal cells were co-treated with glutamate and/or different concentrations of zileuton (1, 2, 5, 10, 50, and 100 µM). As shown in Fig. 1, zileuton decreased HT22 cell death induced by glutamate in dose-dependently. The lowest concentration at which zileuton conferred maximum neuroprotection was 10 µM. Zileuton had no effect on survival of the control cells without glutamate treatment. Based on the results, zileuton at the concentration of 10 µM was used for the following experiments.
HT22 cells were exposed to 5 mM glutamate with or without zileuton at the concentrations of 1, 2, 5, 10, 50 or 100 µM. Cell survival was measured with MTT assay at 24 h after glutamate treatment. Control cells were treated with 10 µM zileuton only. ** p<0.01, compared to cells treated with glutamate alone (Glu), n=4.
Glutamate induced cell death in HT22 cells is mainly mediated by the increase in ROS production. It is reported that glutamate significantly increases ROS production in HT22 cells at 8–9 h after treatment, which is followed by a second burst approximately at 10 h after glutamate treatment.15) 12,15-LOXs are thought to be responsible for the second burst of ROS generation.15) We examined the effects of zileuton on ROS production at 12 h after glutamate exposure using a cell-permeable fluorescent ROS probe (carboxy-H2DCFDA). Compared to the vehicle-treated group, zileuton did not change basal ROS levels. Glutamate markedly increased intracellular ROS levels, which was remarkably reduced by zileuton co-treatment (Fig. 2, representative images from 3 indepednent experiments). Our observation that zileuton prevented glutamate-induced ROS production suggested that, in addition to 12,15-LOXs, 5-LOX also contributed to ROS production induced by glutamate oxidative stress.
HT22 cells were treated with glutamate and zileuton (10 µM) for 12 h followed by incubation with a ROS fluorescent probe H2DCFDA-DA (5 µM) for 10 min. The cells were photographed under light microscopy (A) or fluorescence microscopy (B).
We and others have reported that exposure of HT22 cells to glutamate results in GSH depletion,21) which at least in part is responsible for elevated ROS production in HT22 cells following glutamate treatment. Thus, we further investigated if zileuton restored glutamate-impaired intracellular GSH levels. Consistent with previous reports, glutamate significantly reduced cellular GSH levels at 12 h post-glutamate treatment. However, at the protective dose (10 µM), zileuton did not prevent glutamate-induced depletion of intracellular GSH levels (Fig. 3A). The results suggest that zileuton did not protect neurons from oxidative stress by restoring GSH levels.
(A) HT22 cells were treated with glutamate with or without zileuton (10 µM). GSH concentration was determined 12 h later. * p<0.05 compared to cells treated with glutamate alone (Glu), n=3. (B) HT22 cells were treated with glutamate with or without zileuton (10 µM) and ZVAD-fmk (10 µM). Cell viability was determined with MTT assay 24 h later. ** p<0.01 compared to cells treated with glutamate alone (Glu), n=4.
Stroke induced cell death in a caspase-dependet manner22) and zileuton has been reported to reduce cell death following stroke.23) Moreover, apoptosis is reported to be a component of HT22 cell death induced by glutamate.15,24) Thus, we investigated whether zileuton prevented glutamate oxidative toxicity through a caspase-dependent apoptotic mechanism. ZVAD-fmk is a cell-permeable irreversible inhibitor of caspases. As shown in Fig. 3B, ZVAD-fmk did not protect HT22 cells from glutamate-induced cell death, which was consistent with a previous report.20) Moreover, ZVAD-fmk had no impact on zileuton neuroprotection against glutamate-induced HT22 cell death (Fig. 3B). Collectively, these results suggest that caspase-dependent apoptosis did not contribute to glutamate-induced HT22 cell death and zileuton did not protect neurons against oxidative toxicity by inhibiting caspase-dependent apoptosis.
(A) Zileuton protected HT22 cells against erastin-induced ferroptosis dose-dependently. ** p<0.01 versus cells treated with erastin alone, n=4. (B) The ferroptosis inhibitor ferrostatin-1 reduced glutamate-induced cell death. However, ferrostatin-1 did not synergize with zileuton to protect HT22 cells from glutamate-induced cell death. * p<0.05 and ** p<0.01 versus cells treated with glutamate alone (Glu), n=4. (C) Erastin treatment reduced cellular levels of GSH, which was not prevented by zileuton. (D) Free radical scavenging activity of zileuton was monitored by changes in absorbance at 517 nm of the stable radical DPPH in a cell free system. Zileuton displayed free radical scanvenging activity at 50 and 100 µM but not at 10 µM.
Emerging evidence suggests that glutamate-induced HT22 cell death is a combination of apoptosis, necrosis and ferroptosis. Particularly, ferroptosis, a new, iron-dependent form of programmed cell death, is reported to contribute to glutamate-induced toxicity in neurons.15,19) Thus, we investigated if zileuton protected neurons against glutamate oxidative toxicity through a ferroptosis-dependent mechanism. To the end, we first investigated if zileuton protected HT22 neurons from ferroptotic cell death. HT22 cells were incubated with a ferroptosis-inducing agent erastin (500 nM) in the presence or absence of zileuton. Significant cell death was induced at 18 h after erastin incubation, which was inhibited in a dose-dependent manner by zileuton (Fig. 4A). These results suggest that zileuton protected HT22 neurons from ferroptotic cell death. Moreover, inhibition of ferroptosis with ferrostatin-1 suppressed glutamate-induced cell death in HT22 cells (Fig. 4B). However, zileuton and ferrostatin-1 did not display synergetic protective effects on glutamate-induced HT22 cell death (Fig. 4B). Taken together, these results suggested that suppressing ferroptosis was an important mechanism underlying neuroprotection conferred by zileuton against glutamate oxidative toxicity.
We further investigated the anti-ferroptosis mechanisms of zileuton. It was reported that erastin induces ferroptosis by reducing cellular GSH levels.19) Consistently, we observed that cellular GSH levels were remarkably reduced in HT22 cells at 12 h after exposure to 500 nM erastin, yet zileuton did not elevate erastin-reduced GSH level (Fig. 4C). Ferrostatin-1 protects against ferroptosis by acting as a free-radical scavenger to prevent accumulation of cytosolic and lipid ROS induced by eastin.19) We examined if zileuton exerted free-radical scavenging activity by assessing its activity to oxidize the stable radical DPPH under the cell-free condition. As shown in Fig. 4D, zileuton displayed free-radical scavenging activity only at 50 and 100 µM but not at 10 µM.
We reported two major findings in the study. First, zileuton, a well-established 5-LOX inhibitor, conferred neuroprotection against glutamate oxidative toxicity, suggestive of the involvement of 5-LOX in neuronal death induced by glutamate oxidative stress. Second, we provided evidence that zileuton protected neurons against oxidative toxicity by suppressing ferroptosis, a newly identified form of programmed cell death.
In the model of neuronal death induced by glutamate oxidative toxicity, excessive glutamate inhibits cystine uptake and consequently results in GSH depletion.25) GSH depletion further activates 12,15-LOX, which leads to lipid oxidation and subsequent mitochondrial damage that triggers apoptotic pathways.1) 12,15-LOXs have been shown to contribute to glutamate induced oxidative toxicity in neurons and inhibition of 12,15-LOXs is reported to confer direct neuroprotection against neuronal death induced by glutamate toxicity.7,8) However, it is currently unclear whether 5-LOX also contributes to glutamate induced oxidative toxicity in neurons. Zileuton is a well-established potent 5-LOX inhibitor with IC50 less than 1 µM with high specificity toward 5-LOX.26) In this study, we observed that zileuton at 10 µM significantly ameliorated glutamate oxidative toxicity. Since even at high concentrations (up to 100 µM) zileuton displays very little or no inhibition on related enzymes, including 12,15-LOXs,26) we think that zileuton acted through 5-LOX inhibition rather than 12,15-LOX inhibition to protect neurons from oxidative stress. Thus, our results suggested that, in addition to 12,15-LOXs, 5-LOX also contributed to glutamate-induced oxidative toxicity in neurons. Further studies are needed to investigate if 5-LOX is activated in response to glutamate oxidative stress and if the gene manipulation approach that reduces 5-LOX expression or activity confers neuroprotection against glutamate oxidative toxicity. 5-LOX inhibitors have been reported to protect against cerebral ischemia via suppressing post-ischemic neuro-inflammation.9) However, our results presented here suggested that, in addition to immune-suppression, the neuroprotective mechanism may also contribute to the protection of zileuton following experimental stroke.
It is generally accepted that glutamate elicits oxidative stress, resulting in both necrotic and apoptotic cell death in HT22 cell lines. In our study, ZVAD-fmk, a widely used cell permeable pan-caspase inhibitor, neither rescued cell death induced by glutamate nor affected the neuroprotective effects of zileuton. These results were consistent with previous studies showing that glutamate oxidative stress-induced neuronal cell death is caspase-independent. Thus, our results suggested that zileuton did not protect neurons against glutamate oxidative toxicity by suppressing caspase-dependent apoptosis. However, further studies are needed to investigate if zileuton protects neurons from glutamate oxidative toxicity by inhibiting caspase-independent apoptosis, such as apoptosis induced by translocation of apoptosis-inducing factor.27) Furthermore, recent studies indicated that inhibiting caspase by ZVAD-fmk switched cell death from apoptosis to necroptosis, a newly identified programmed necrosis.16) Our results showed that zileuton rescued HT22 neuronal cells from glutamate oxidative toxicity in the presence of ZVAD-fmk; raising the possibility that zileuton confers neuroprotection by inhibiting necroptosis. Thus, further studies are needed to explore the possibility.
GSH is one of the most abundant antioxidant molecules in the brain and plays a crucial role in mental disorders. GSH depletion and consequent ROS accumulation are thought to be major mechanisms underlying glutamate oxidative toxicity.17,28) We observed that zileuton reduced glutamate-induced ROS levels. However, zileuton did not restore glutamate-depleted GSH, suggesting that the beneficial impact of zileuton was not mediated by restoration of GSH levels. Consistently, other neuroprotectants, such as melatonin, have also been shown to protect neurons from glutamate oxidative toxicity without preventing GSH depletion induced by glutamate.29)
A new finding of our study is that the neuroprotective effects conferred by zileuton involved an anti-ferroptosis mechanism. Ferroptosis is a newly identified iron-dependent form of cell death, which is characterized by the accumulation of lethal levels of lipid peroxidation.15) Iron accelerates free radical reaction and consequently enhances lipid peroxidation that leads to cell death. Recently, it was reported that ferroptosis was involved in the death cascade induced by glutamate oxidative stress.15) In support of the report, we observed that glutamate-induced HT22 cell death was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. Moreover, zileuton protected HT22 neuronal cells from erastin-induced ferroptosis. However, zileuton and ferrostatin-1 did not display synergetic protective effects on glutamate-induced HT22 cell death, implying that zileuton and ferrostatin-1 acted through the same pathway to protect against glutamate oxidative stress. Taken together, our results supported that zileuton protected neurons from glutamate-induced oxidative stress at least in part by suppressing ferroptosis.
Erastin induces ferroptosis by decreasing cellular GSH levels.19) Our results suggested that zileuton did not prevent erastin depletion of cellular GSH. Moreover, the anti-ferroptosis agent ferrostatin-1 has been reported to act through the free-radical scavenging mechanism to protect against ferroptosis.19) We observed that zileuton displayed free-radical scavenging activity only at high doses (50 and 100 µM), but not at 10 µM. Thus, at least at the low protective doses, zileuton did not act through the free radical scavenging mechanism to protect against ferroptosis. Further studies are needed to investigate whether zileuton protects against ferroptosis by inhibiting 5-LOX.
In summary, the 5-LOX inhibitor zileuton conferred direct neuroprotection against glutamate-induced oxidative cell death. Moreover, we provided evidence that suppressing ferroptosis was a novel mechanism underlying neuroprotective effects of zileuton against glutamate oxidative stress.
The project is supported by the Grants from National Science Foundation of China (81371278, 81471336, 81171246), the Priority Academic Program Development of Jiangsu Higher Education Institutions of China (PAPD) and funding from BM2013003.
The authors declare no conflict of interest.
