Food & Function
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Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: P. Chen, Y. Ho, M. Wu, S. Huang, P. Chen, M. Tai, C. Ho and J. Yen, Food Funct., 2014, DOI: 10.1039/C4FO00948G.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/foodfunction
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Food & Function View Article Online
DOI: 10.1039/C4FO00948G
Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells Pei-Yi Chen a‡, Yi-Ru Hob‡, Ming-Jiuan Wuc, Shun-Ping Huangb, Po-Kong Chenb, Mi-Hsueh Taib, ChiTang Hod, Jui-Hung Yenb*
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Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X DOI: 10.1039/b000000x Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells in both cobalt chloride (CoCl2)- and low oxygeninduced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl2-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxiainducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl2 exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3K/Akt-dependent signaling pathways in PC12 cells.
1.
INTRODUCTION
Hypoxia is a pathological state in which the oxygen concentration drops to a level insufficient to support normal metabolism. Neurons are the cells most sensitive to hypoxia, which has been reported to disturb brain function and can lead to a variety of neurological disorders such as motor dysfunction, learning disabilities, epilepsy, seizure and dementia1,2,3. Evidence has implicated that prolonged hypoxia causes neuron necrosis and apoptosis in the brain4. Hypoxia is involved in the development of neurodegenerative diseases. It facilitates the pathogenesis of Alzheimer’s disease through Aβ accumulation and tau phosphorylation and by promoting neuron degeneration5,6. Cells have developed a programmed defense response against hypoxia7. Hypoxia-inducible factor 1 (HIF-1) is a major transcription factor involved in the cell responses to hypoxic stress. HIF-1 consists of a hypoxia inducible subunit HIF-1α and a constitutively expressed subunit HIF-1β. In the presence of oxygen, HIF-1α binds pVHL (Von Hippel–Lindau tumor suppressor) through 2 hydroxylated proline residues. The binding of pVHL leads to the ubiquitination of HIF-1α, which targets it to the proteasome for degradation8. In contrast, in response to hypoxia, proline hydroxylation is inhibited. pVHL is no longer able to bind and target HIF-1α for proteasomal degradation, which leads to HIF-1α accumulation and translocation to the nucleus. There, HIF-1α dimerizes with HIF-1β and specifically binds to the hypoxia response element (HRE) of the promoters of target genes involved in erythropoiesis, glycolysis, angiogenesis
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and cell survival9,10,11. Furthermore, it has also been shown that HIF-1α plays an essential role in neuroprotection and maintaining the integrity of the brain under hypoxic and ischemic conditions in animal models12. Flavonoids, the most common group of polyphenolic compounds in the human diet, are abundant in fruits and vegetables13. They are believed to have beneficial effects against multiple diseases, including cancers, cardiovascular disease and inflammatory and neurodegenerative disorders14,15. Previous studies have revealed that flavonoid compounds could cross the blood-brain barrier (BBB) and might possess a neuroprotection ability16. Fisetin (3,7,3',4'-tetrahydroxyflavone) (supplemental Fig. 1) belongs to the flavonol subgroup of flavonoids found in many vegetables and fruit and is especially rich in apples, strawberries, onions and mangoes. It exhibits antioxidant, antiinflammatory and anti-carcinogenic activities17,18,19. Recently, it was also considered to possess neuroprotective effects against the aging process, cerebral damage and neurodegenerative disorders20,21. It has been shown to reduce excessive formation of the Aβ protein, which is associated with Alzheimer’s disease22. In addition to neuroprotection, fisetin could promote neuronal differentiation and ERK-dependent long-term potentiation, maintain cognitive function and enhance memory in an animal model23,24. Taken together, these studies reveal that fisetin is a potential cytoprotective compound for neuronal cells. However, the cytoprotective effect of fisetin on neuronal cells exposed to hypoxic conditions is yet unknown. It has been reported that cobalt chloride (CoCl2) treatment could mimic some of the hypoxic responses observed in cultured
Food & Function Accepted Manuscript
Published on 14 November 2014. Downloaded by Clemson University on 21/11/2014 11:32:37.
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cells, including the generation of reactive oxygen species (ROS) and induction of transcriptional changes of genes, such as HIF1α, erythropoietin and glycolytic enzymes. In addition, CoCl2dependent modulation of gene products in hypoxic and DNA damage responses were also observed25, 26. Therefore, CoCl2 has been used as a reagent in both in vitro and in vivo to mimic hypoxic responses27,28,29. The PC12 cell line is a rat pheochromocytoma cell line and is a useful model for studying cell survival, cell differentiation and oxygen-sensitive molecular and cellular mechanisms of neuronal cells30,31. In the present study, we aim to investigate the cytoprotective effect and underlying mechanisms of fisetin against hypoxia-induced cell death in PC12 cells.
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2.5 Reverse transcription quantitative PCR (RT-Q-PCR) analysis
Materials and Methods
2.1 Materials Fisetin, cobalt chloride (CoCl2), RPMI-1640 medium, nonessential amino acid (NEAA), poly-L-lysine, dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise indicated. Inhibitors U0126, SB203580 and LY294002 were purchased from Promega (Madison, WI, USA). SP600125 was purchased from Enzo Life Sciences (Ann Arbor, MI, USA). The mouse 7S nerve growth factor (NGF) was purchased from Millipore (Billerica, MA, USA).
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2.2 Cell culture
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The PC12 cells were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum (HS) (Invitrogen, Carlsbad, CA, USA), 5% fetal bovine serum (FBS) (Biological Industries, Kibbutz Haemek, Israel) and 1% non-essential amino acid (NEAA). The cells were cultured in a 5% CO2 incubator at 37°C. For experiments using a hypoxia incubator, the cells were cultured in 1% O2, 5% CO2 and 94% N2 at 37°C. For analysis of differentiated PC12 cells, the cells were seeded on poly-L-lysine-coated 6-well plates and incubated in RPMI medium with 10% HS, 5% FBS and 1% NEAA for 24 h. The medium was replaced with fresh medium with the addition of nerve growth factor (NGF; 50 ng/ml) for an additional 72 h incubation.
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2.3 Compound treatments and analysis of cell viability
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The PC12 cells (1x106/ml) were pre-treated with the indicated reagent or vehicle (0.1% DMSO) for 1 h before exposure of CoCl2. Cell viability was determined through the reduction of MTT to purple formazan crystal by mitochondria. The cells were incubated with a MTT solution (1 mg/ml) for 3 h at 37°C. The crystals were dissolved in DMSO, and the extent of the reduction of MTT was determined as the absorbance at 550 nm32. For the N-acetyl-L-cysteine (NAC) treatment experiment, the cell viability was measured with a Calcein AM (Invitrogen) assay. PC12 cells were incubated with Calcein AM dye (1 µM) for 30 min at 37°C. The fluorescence intensity was determined using a 485 nm excitation and 530 nm emission33. 2.4 Analysis of reactive oxygen species (ROS) generation
PC12 cells (1x106/ml) were seeded on poly-L-lysine-coated 6well plates and incubated in RPMI medium (10% HS, 5% FBS and 1%NEAA) for 24 h. Then, the medium was replaced with fresh serum-free RPMI medium, H2DCFDA (5 µM) was added and the cells were incubated at 37°C in the dark. After 30 min incubation, the PC12 cells were treated with fisetin (40 µM) for 1 h prior to CoCl2 (200 µM) exposure. After 1 h incubation, the medium was removed and the cells were harvested for ROS analysis. Three independent samples of 10,000 cells were analyzed via flow cytometry using the FL1 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). The data were expressed as the relative percentage of the geometric mean fluorescence intensity.
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PC12 cells (1x106/ml) were seeded on 60 mm dishes in RPMI medium (10% HS, 5% FBS and 1% NEAA) and treated with vehicle (0.1% DMSO) or the indicated compounds for the indicated periods. Total cellular RNA was prepared using the Total RNA Mini Kit (Geneaid, Taipei, Taiwan). The reverse transcription of RNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed in a reaction mixture containing cDNA, specific primers [HIF-1α, 5'CGAGCTGCCTCTTCGACAAG-3' (forward) and 5'CCCAGCCGCTGGAGCTA-3' (reverse); β-actin, 5'CCTCTGAACCCTAAGGCCAA-3' (forward) and 5'AGCCTGGATGGCTACGTACA-3' (reverse)] and Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Burlington, CA). The amplification was performed in a Roche Light Cycler 480 sequence detection system. The PCR conditions used are as follows: 50°C for 2 min, 94°C for 4 min, 45 cycles at 94°C for 60 sec, 58°C for 60 sec and 72°C for 60 sec. The ∆∆Ct method was used for the analysis of HIF-1α mRNA expression, estimated in triplicate samples and normalized to β-actin expression levels. 2.6 Western blot analysis PC12 cells (1 x 106/ml) were seeded on 100 mm dishes in RPMI medium (10% HS, 5% FBS and 1% NEAA) and subsequently incubated with the indicated agents for the indicated time periods. Total cellular proteins were prepared using RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL). For nuclear and cytosolic protein preparation, the cells were harvested using the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein preparation was separated on 10% SDS-PAGE and was subsequently transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA). The blots were incubated with the following specific antibodies: anti-p44/42 MAPK(Erk1/2), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti-p38, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-Akt, antiphospho-Akt (Ser-147) (Cell Signaling Technology, Inc.), antiHIF-1α, anti-HDAC2 (GeneTex, Irvine, CA, USA) and monoclonal anti-β-actin (Sigma-Aldrich). The blots were incubated with the appropriate HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Proteins were detected using Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer), and the signal
Food & Function Accepted Manuscript
DOI: 10.1039/C4FO00948G
Page 3 of 10
Food & Function View Article Online
was visualized with Amersham Hyperfilm™ Healthcare, Buckinghamshire, UK).
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2.7 Transfection and luciferase reporter assay
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PC12 cells (1x106/ml) were seeded on poly-L-lysine-coated 24wells plates in DMEM medium (10% HS and 5% FBS) for 24 h. The cells were transfected with the pGL4.42[Luc2P/HRE/Hygro] plasmid and the Renilla luciferase control vector (Promega) (plasmid ratio, 20:1) using Lipofectamine 2000 (Invitrogen). After 24 h transfection, the cells were treated with the indicated agents for an additional 24 h. The cells were harvested, and the luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The luciferase intensities were normalized to the activity of the Renilla luciferase.
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2.8 Transfection of small interference RNA (siRNA) 70 15
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PC12 cells (1x106/ml) were seeded onto poly-L-lysine-coated 6well plates in DMEM medium (10% HS and 5% FBS) for 24 h. Then, the cells were incubated with serum-free OPTI-MEM medium and transfected with the negative control (si-NC) or ratspecific HIF-1α siRNA (si-HIF-1α) duplexes [5'GAAGGAACCUGAUGCUUUAACUU-3' (forward) and 5'AAGUUAAAGCAUCAGGUUCCUUC-3' (reverse)]34 (GeneDireX Inc., Las Vegas, NV, USA) at a final concentration of 150 pmol/ml using Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h transfection, the cells were pre-treated with the vehicle or fisetin (40 µM) for 1 h and then exposed to CoCl2 (200 µM) for an additional 24 h. The cell viability was measured with the MTT assay.
viability was markedly increased from 51.7±1.0% to 65.0±1.1% and 75.2±3.1%, respectively (Fig. 1D). These data confirm that fisetin restores PC12 cell viability and protects against hypoxiainduced cell death. It has been shown that CoCl2 also causes cytotoxicity in NGFdifferentiated PC12 cells35. We therefore investigated the effect of fisetin on differentiated PC12 cells exposed to CoCl2. The PC12 cells were differentiated by incubating with 50 ng/ml nerve growth factor (NGF) for 72 h and then treated with vehicle (0.1% DMSO) or fisetin (40 and 60 µM) for 1 h followed by exposure to CoCl2 (200 µM) for an additional 24 h. As shown in Figure 1E, cell viabilities were significantly increased by fisetin (40 and 60 µM) from 52.9±0.2% to 80.4±1.6% and 84.0±6.9%, respectively. These results reveal that fisetin provides cytoprotection against CoCl2 toxicity in both undifferentiated and differentiated PC12 cells.
2.9 Statistical Analysis
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All experiments were repeated at least three times. All values are expressed as the mean ± SD. The results were analyzed using a One-way ANOVA with a Dunnet’s post-hoc test, and a p-value of < 0.05 was considered statistically significant.
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Results
3.1 Fisetin protects PC12 cells from hypoxia-induced cell death Cobalt chloride (CoCl2) is widely used as a reagent to mimic hypoxia in PC12 cells. To confirm the cytotoxic effect of CoCl2 on PC12 cells, the cells were treated with CoCl2 (0-250 µM) for 24 h and the cell viability was measured with an MTT assay. Figure 1A and 1B confirm that 24 h CoCl2 (0-250 µM) treatment in PC12 cells caused a dose-dependent cytotoxicity. CoCl2 caused cell shrinkage and approximately 55% cell death at a concentration of 200 µM. To examine the protective effect of fisetin in CoCl2-treated PC12 cells, cells were treated with vehicle (0.1% DMSO) or fisetin (0-60 µM) for 1 h before exposure to 200 µM CoCl2 for 24 h. Figure 1C shows that fisetin (5-60 µM) significantly increased the cell viability in a dosedependent manner up to 90.6±8.9% (p
View Article Online View Journal
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: P. Chen, Y. Ho, M. Wu, S. Huang, P. Chen, M. Tai, C. Ho and J. Yen, Food Funct., 2014, DOI: 10.1039/C4FO00948G.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/foodfunction
Page 1 of 10
Food & Function View Article Online
DOI: 10.1039/C4FO00948G
Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells Pei-Yi Chen a‡, Yi-Ru Hob‡, Ming-Jiuan Wuc, Shun-Ping Huangb, Po-Kong Chenb, Mi-Hsueh Taib, ChiTang Hod, Jui-Hung Yenb*
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Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X DOI: 10.1039/b000000x Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells in both cobalt chloride (CoCl2)- and low oxygeninduced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl2-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxiainducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl2 exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3K/Akt-dependent signaling pathways in PC12 cells.
1.
INTRODUCTION
Hypoxia is a pathological state in which the oxygen concentration drops to a level insufficient to support normal metabolism. Neurons are the cells most sensitive to hypoxia, which has been reported to disturb brain function and can lead to a variety of neurological disorders such as motor dysfunction, learning disabilities, epilepsy, seizure and dementia1,2,3. Evidence has implicated that prolonged hypoxia causes neuron necrosis and apoptosis in the brain4. Hypoxia is involved in the development of neurodegenerative diseases. It facilitates the pathogenesis of Alzheimer’s disease through Aβ accumulation and tau phosphorylation and by promoting neuron degeneration5,6. Cells have developed a programmed defense response against hypoxia7. Hypoxia-inducible factor 1 (HIF-1) is a major transcription factor involved in the cell responses to hypoxic stress. HIF-1 consists of a hypoxia inducible subunit HIF-1α and a constitutively expressed subunit HIF-1β. In the presence of oxygen, HIF-1α binds pVHL (Von Hippel–Lindau tumor suppressor) through 2 hydroxylated proline residues. The binding of pVHL leads to the ubiquitination of HIF-1α, which targets it to the proteasome for degradation8. In contrast, in response to hypoxia, proline hydroxylation is inhibited. pVHL is no longer able to bind and target HIF-1α for proteasomal degradation, which leads to HIF-1α accumulation and translocation to the nucleus. There, HIF-1α dimerizes with HIF-1β and specifically binds to the hypoxia response element (HRE) of the promoters of target genes involved in erythropoiesis, glycolysis, angiogenesis
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and cell survival9,10,11. Furthermore, it has also been shown that HIF-1α plays an essential role in neuroprotection and maintaining the integrity of the brain under hypoxic and ischemic conditions in animal models12. Flavonoids, the most common group of polyphenolic compounds in the human diet, are abundant in fruits and vegetables13. They are believed to have beneficial effects against multiple diseases, including cancers, cardiovascular disease and inflammatory and neurodegenerative disorders14,15. Previous studies have revealed that flavonoid compounds could cross the blood-brain barrier (BBB) and might possess a neuroprotection ability16. Fisetin (3,7,3',4'-tetrahydroxyflavone) (supplemental Fig. 1) belongs to the flavonol subgroup of flavonoids found in many vegetables and fruit and is especially rich in apples, strawberries, onions and mangoes. It exhibits antioxidant, antiinflammatory and anti-carcinogenic activities17,18,19. Recently, it was also considered to possess neuroprotective effects against the aging process, cerebral damage and neurodegenerative disorders20,21. It has been shown to reduce excessive formation of the Aβ protein, which is associated with Alzheimer’s disease22. In addition to neuroprotection, fisetin could promote neuronal differentiation and ERK-dependent long-term potentiation, maintain cognitive function and enhance memory in an animal model23,24. Taken together, these studies reveal that fisetin is a potential cytoprotective compound for neuronal cells. However, the cytoprotective effect of fisetin on neuronal cells exposed to hypoxic conditions is yet unknown. It has been reported that cobalt chloride (CoCl2) treatment could mimic some of the hypoxic responses observed in cultured
Food & Function Accepted Manuscript
Published on 14 November 2014. Downloaded by Clemson University on 21/11/2014 11:32:37.
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Food & Function
Page 2 of 10 View Article Online
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Published on 14 November 2014. Downloaded by Clemson University on 21/11/2014 11:32:37.
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cells, including the generation of reactive oxygen species (ROS) and induction of transcriptional changes of genes, such as HIF1α, erythropoietin and glycolytic enzymes. In addition, CoCl2dependent modulation of gene products in hypoxic and DNA damage responses were also observed25, 26. Therefore, CoCl2 has been used as a reagent in both in vitro and in vivo to mimic hypoxic responses27,28,29. The PC12 cell line is a rat pheochromocytoma cell line and is a useful model for studying cell survival, cell differentiation and oxygen-sensitive molecular and cellular mechanisms of neuronal cells30,31. In the present study, we aim to investigate the cytoprotective effect and underlying mechanisms of fisetin against hypoxia-induced cell death in PC12 cells.
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2.5 Reverse transcription quantitative PCR (RT-Q-PCR) analysis
Materials and Methods
2.1 Materials Fisetin, cobalt chloride (CoCl2), RPMI-1640 medium, nonessential amino acid (NEAA), poly-L-lysine, dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise indicated. Inhibitors U0126, SB203580 and LY294002 were purchased from Promega (Madison, WI, USA). SP600125 was purchased from Enzo Life Sciences (Ann Arbor, MI, USA). The mouse 7S nerve growth factor (NGF) was purchased from Millipore (Billerica, MA, USA).
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The PC12 cells were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum (HS) (Invitrogen, Carlsbad, CA, USA), 5% fetal bovine serum (FBS) (Biological Industries, Kibbutz Haemek, Israel) and 1% non-essential amino acid (NEAA). The cells were cultured in a 5% CO2 incubator at 37°C. For experiments using a hypoxia incubator, the cells were cultured in 1% O2, 5% CO2 and 94% N2 at 37°C. For analysis of differentiated PC12 cells, the cells were seeded on poly-L-lysine-coated 6-well plates and incubated in RPMI medium with 10% HS, 5% FBS and 1% NEAA for 24 h. The medium was replaced with fresh medium with the addition of nerve growth factor (NGF; 50 ng/ml) for an additional 72 h incubation.
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2.3 Compound treatments and analysis of cell viability
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The PC12 cells (1x106/ml) were pre-treated with the indicated reagent or vehicle (0.1% DMSO) for 1 h before exposure of CoCl2. Cell viability was determined through the reduction of MTT to purple formazan crystal by mitochondria. The cells were incubated with a MTT solution (1 mg/ml) for 3 h at 37°C. The crystals were dissolved in DMSO, and the extent of the reduction of MTT was determined as the absorbance at 550 nm32. For the N-acetyl-L-cysteine (NAC) treatment experiment, the cell viability was measured with a Calcein AM (Invitrogen) assay. PC12 cells were incubated with Calcein AM dye (1 µM) for 30 min at 37°C. The fluorescence intensity was determined using a 485 nm excitation and 530 nm emission33. 2.4 Analysis of reactive oxygen species (ROS) generation
PC12 cells (1x106/ml) were seeded on poly-L-lysine-coated 6well plates and incubated in RPMI medium (10% HS, 5% FBS and 1%NEAA) for 24 h. Then, the medium was replaced with fresh serum-free RPMI medium, H2DCFDA (5 µM) was added and the cells were incubated at 37°C in the dark. After 30 min incubation, the PC12 cells were treated with fisetin (40 µM) for 1 h prior to CoCl2 (200 µM) exposure. After 1 h incubation, the medium was removed and the cells were harvested for ROS analysis. Three independent samples of 10,000 cells were analyzed via flow cytometry using the FL1 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). The data were expressed as the relative percentage of the geometric mean fluorescence intensity.
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PC12 cells (1x106/ml) were seeded on 60 mm dishes in RPMI medium (10% HS, 5% FBS and 1% NEAA) and treated with vehicle (0.1% DMSO) or the indicated compounds for the indicated periods. Total cellular RNA was prepared using the Total RNA Mini Kit (Geneaid, Taipei, Taiwan). The reverse transcription of RNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed in a reaction mixture containing cDNA, specific primers [HIF-1α, 5'CGAGCTGCCTCTTCGACAAG-3' (forward) and 5'CCCAGCCGCTGGAGCTA-3' (reverse); β-actin, 5'CCTCTGAACCCTAAGGCCAA-3' (forward) and 5'AGCCTGGATGGCTACGTACA-3' (reverse)] and Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Burlington, CA). The amplification was performed in a Roche Light Cycler 480 sequence detection system. The PCR conditions used are as follows: 50°C for 2 min, 94°C for 4 min, 45 cycles at 94°C for 60 sec, 58°C for 60 sec and 72°C for 60 sec. The ∆∆Ct method was used for the analysis of HIF-1α mRNA expression, estimated in triplicate samples and normalized to β-actin expression levels. 2.6 Western blot analysis PC12 cells (1 x 106/ml) were seeded on 100 mm dishes in RPMI medium (10% HS, 5% FBS and 1% NEAA) and subsequently incubated with the indicated agents for the indicated time periods. Total cellular proteins were prepared using RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL). For nuclear and cytosolic protein preparation, the cells were harvested using the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein preparation was separated on 10% SDS-PAGE and was subsequently transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA). The blots were incubated with the following specific antibodies: anti-p44/42 MAPK(Erk1/2), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti-p38, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-Akt, antiphospho-Akt (Ser-147) (Cell Signaling Technology, Inc.), antiHIF-1α, anti-HDAC2 (GeneTex, Irvine, CA, USA) and monoclonal anti-β-actin (Sigma-Aldrich). The blots were incubated with the appropriate HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Proteins were detected using Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer), and the signal
Food & Function Accepted Manuscript
DOI: 10.1039/C4FO00948G
Page 3 of 10
Food & Function View Article Online
was visualized with Amersham Hyperfilm™ Healthcare, Buckinghamshire, UK).
ECL (GE
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2.7 Transfection and luciferase reporter assay
Published on 14 November 2014. Downloaded by Clemson University on 21/11/2014 11:32:37.
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10
PC12 cells (1x106/ml) were seeded on poly-L-lysine-coated 24wells plates in DMEM medium (10% HS and 5% FBS) for 24 h. The cells were transfected with the pGL4.42[Luc2P/HRE/Hygro] plasmid and the Renilla luciferase control vector (Promega) (plasmid ratio, 20:1) using Lipofectamine 2000 (Invitrogen). After 24 h transfection, the cells were treated with the indicated agents for an additional 24 h. The cells were harvested, and the luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The luciferase intensities were normalized to the activity of the Renilla luciferase.
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PC12 cells (1x106/ml) were seeded onto poly-L-lysine-coated 6well plates in DMEM medium (10% HS and 5% FBS) for 24 h. Then, the cells were incubated with serum-free OPTI-MEM medium and transfected with the negative control (si-NC) or ratspecific HIF-1α siRNA (si-HIF-1α) duplexes [5'GAAGGAACCUGAUGCUUUAACUU-3' (forward) and 5'AAGUUAAAGCAUCAGGUUCCUUC-3' (reverse)]34 (GeneDireX Inc., Las Vegas, NV, USA) at a final concentration of 150 pmol/ml using Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h transfection, the cells were pre-treated with the vehicle or fisetin (40 µM) for 1 h and then exposed to CoCl2 (200 µM) for an additional 24 h. The cell viability was measured with the MTT assay.
viability was markedly increased from 51.7±1.0% to 65.0±1.1% and 75.2±3.1%, respectively (Fig. 1D). These data confirm that fisetin restores PC12 cell viability and protects against hypoxiainduced cell death. It has been shown that CoCl2 also causes cytotoxicity in NGFdifferentiated PC12 cells35. We therefore investigated the effect of fisetin on differentiated PC12 cells exposed to CoCl2. The PC12 cells were differentiated by incubating with 50 ng/ml nerve growth factor (NGF) for 72 h and then treated with vehicle (0.1% DMSO) or fisetin (40 and 60 µM) for 1 h followed by exposure to CoCl2 (200 µM) for an additional 24 h. As shown in Figure 1E, cell viabilities were significantly increased by fisetin (40 and 60 µM) from 52.9±0.2% to 80.4±1.6% and 84.0±6.9%, respectively. These results reveal that fisetin provides cytoprotection against CoCl2 toxicity in both undifferentiated and differentiated PC12 cells.
2.9 Statistical Analysis
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All experiments were repeated at least three times. All values are expressed as the mean ± SD. The results were analyzed using a One-way ANOVA with a Dunnet’s post-hoc test, and a p-value of < 0.05 was considered statistically significant.
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Results
3.1 Fisetin protects PC12 cells from hypoxia-induced cell death Cobalt chloride (CoCl2) is widely used as a reagent to mimic hypoxia in PC12 cells. To confirm the cytotoxic effect of CoCl2 on PC12 cells, the cells were treated with CoCl2 (0-250 µM) for 24 h and the cell viability was measured with an MTT assay. Figure 1A and 1B confirm that 24 h CoCl2 (0-250 µM) treatment in PC12 cells caused a dose-dependent cytotoxicity. CoCl2 caused cell shrinkage and approximately 55% cell death at a concentration of 200 µM. To examine the protective effect of fisetin in CoCl2-treated PC12 cells, cells were treated with vehicle (0.1% DMSO) or fisetin (0-60 µM) for 1 h before exposure to 200 µM CoCl2 for 24 h. Figure 1C shows that fisetin (5-60 µM) significantly increased the cell viability in a dosedependent manner up to 90.6±8.9% (p
