Tetrachlorobenzoquinone-induced Nrf2 Confers Neuron-Like PC12

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Tetrachlorobenzoquinone-induced Nrf2 Confers Neuron-Like PC12 Cells Resistance to Endoplasmic Reticulum Stress via Regulating Glutathione Synthesis and Protein Thiol Homeostasis Zixuan Liu, Wenjing Dong, Bingwei Yang, Lu Peng, Xiaomin Xia, Lanxiang Pu, Na Zhang, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00209 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Chemical Research in Toxicology

Tetrachlorobenzoquinone-induced Nrf2 Confers Neuron-Like PC12 Cells Resistance to Endoplasmic Reticulum Stress via Regulating Glutathione Synthesis and Protein Thiol Homeostasis Zixuan Liu, Wenjing Dong, Bingwei Yang, Lu Peng, Xiaomin Xia, Lanxiang Pu, Na Zhang, Erqun Song, Yang Song*

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, People’s Republic of China, 400715

*Corresponding Author: College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing, People’s Republic of China, 400715. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail: [email protected]

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TABLE OF CONTENTS

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ABSTRACT Our previous studies demonstrated that tetrachlorobenzoquinone (TCBQ) is toxic to neuron-like cells, which related to endoplasmic reticulum (ER) stress-induced apoptosis. However, it remains unclear whether TCBQ causes the opening of cellular defense responses. Here we found that activation of nuclear factor erythroid-derived 2-like 2 (Nrf2) triggered an adaptive response against the neurotoxicity induced by TCBQ through the upregulation of intracellular glutathione (GSH) levels in rat pheochromocytoma PC12 cells. TCBQ upregulated the levels of GSH mainly by the following two ways: (i) Nrf2 activation induced the expression of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11, also called xCT); (ii) Nrf2 activation resulted in increased the expression of glutamylcysteine ligase (GCL). GSH is involved in cell antioxidant ability and protein thiol homeostasis, especially in the ER. Therefore, GSH has the ability to inhibit ER stress and promote cell survival. Our data showed that decreasing GSH levels exacerbated TCBQ-induced depletion of protein-SH, particularly in the ER. Conversely, increasing GSH levels attenuated TCBQ-induced protein damage, degree of ER stress, and cell death. These findings demonstrated that GSH-inhibited cells were vulnerable to TCBQ-induced ER stress and apoptosis. Overall, our results analysed the relationships between Nrf2 and ER stress in response to TCBQ and showed that activation of Nrf2-GSH played a protective role against TCBQ-induced ER stress-associated neurotoxicity via regulating GSH synthesis and protein thiol homeostasis.

Keywords Tetrachlorobenzoquinone · Nrf2 · Glutathione · Endoplasmic reticulum stress · protein-SH

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INTRODUCTION It is well known that polyhalogenated quinones is a representative of toxic intermediates, which can cause various hazardous effects in vivo, including cytotoxic and genotoxic effects.1,

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Tetrachlorobenzoquinone (TCBQ), an example of halogenated quinoid compound, has been widely observed as a major reactive metabolite in the oxidation processes of degrading of fungicide hexachlorobenzene (HCB) and pentachlorophenol (PCP).2-6 The molecular mechanisms of TCBQ-induced toxicity have been extensively studied.11-19 Our previous studies confirmed the mechanisms of neurotoxicity induced by TCBQ,13,

15, 16, 18, 19

and highlighted that TCBQ activated

signaling pathways involved in ER stress-associated apoptosis.16,

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The previous experiments

investigated the mechanism by which TCBQ induced neurotoxicity, however, it is still unclear whether the cellular defense system is involved in cell resistance to TCBQ-induced neurotoxicity. Nrf2 is a key oxidative stress-responsive transcription factor directly related to combating xenobiotics and oxidative stress.20,

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Under unstressed conditions, Nrf2 is mainly located in the

cytoplasm and acts as a short-lived protein that primarily bound to Kelch-like ECH-associated protein 1 (Keap1) which associates with Cullin3 to target Nrf2 for ubiquitination and proteasomal degradation.22 Electrophiles and oxidative stress activate Nrf2 by allowing Nrf2 translocate into the nucleus where it binds to antioxidant-response elements (AREs) in the promoters of several cellular defense genes,21 including glutathione biosynthetic enzyme (the modifier (GCLM) and the catalytic (GCLC) subunits of γ-glutamylcysteine ligase (GCL)), the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11, also called xCT), glutathione reductase (GSR), thioredoxin (Trx), GSH-dependent antioxidant enzymes (glutathione S-transferases (GST), glutathione peroxidase 2 (GPX2) and heme 4


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oxygenase-1 (HO-1)).23-25 The activation of Nrf2 by TCBQ suggested that mammalian cells have developed a robust defensive system to alleviate the toxicity of TCBQ. Several human diseases, such as neurodegenerative diseases, are associated with the ER stress and oxidative injury.26 Our work recently highlighted that TCBQ challenge resulted in excessive ER stress depending on the duration of oxidative stress.19 The ER is a specialized cellular compartment for protein folding and processing, which is important for repairing abnormal proteins and very sensitive to variations in redox state. Depeletion of protein thiol groups or formation of abnormal disulfide bonds can disrupt protein thiol (protein-SH) homeostasis. Some studies have shown that intracellular GSH played a crucial role in the defense against cytotoxicity of quinone, especially by preventing quinone form perturbating protein-SH homeostasis.27, 28 GSH provides the main redox buffer for the formation of native disulphide bonds and the isomerization of incorrect disulphide bonds in the ER.29 GSH may play a role in keeping ER oxidoreductases in their reduced form so that reduction or isomerization reactions may operate functionally.30,

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Although Nrf2 is a critical factor for GSH synthesis, there is limited

evidence to show the correlation between Nrf2 and ER stress. In this study, a well-established in vitro experimental model (PC12 cell line) was employed to detect the mechanism of cell resistance to TCBQ insult. We found that the transcription factors Nrf2 generated a cytoprotective effect by increasing the production of GSH. We delineated the inter-relationship between ER stress and Nrf2, which involved GSH production required for maintaining protein-SH homeostasis in the ER during the condition that TCBQ caused protein damage. Our findings therefore complemented the research of TCBQ on the cellular defense system and allowed further insight into the mechanisms of molecular events caused by TCBQ. 5



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MATERIALS AND METHODS Materials and Reagents TCBQ (purity of 98%), N-acetyl-L-cysteine (NAC), glutathione ethyl ester (GSH-E) and buthionine sulfoximine (BSO) were provided by Aladdin Reagent Database Inc. (Shanghai, China). Dibromobimane was obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). The antibodies to GCLM, GCLC and GSR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies to chaperone glucose-related protein 78 (GRP78), GAPDH and Lamin B were obtained from Proteintech group, Inc. (Wuhan, China). The antibodies to p-inositolrequiring kinase/endonuclease 1α (IRE1α) (Ser726), p-protein kinase R-like ER kinase (PERK) (Thr980) and activating transcriptional factor 4 (ATF4) were obtained from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). The antibody to C/EBP-homologous protein (CHOP) was obtained from Ruiying Biological (Suzhou, China). The nuclear/cytoplasmic extraction reagents was supplied by Sangon Biotech Co., Ltd (Shanghai, China). Lactate dehydrogenase (LDH) assay kit and reduced GSH assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibody to Nrf2 and Annexin V-FITC apoptosis detection kit were purchased from Wanlei bio Co., Ltd. (Shenyang, China). 6-Diamidino-2-phenylindole dihydrochloride (DAPI) and BeyoECL Star were purchased from Beyotime Institute of Biotechnology (Nanjing, China). Dulbecco’s modified Eagle's medium (DMEM) and trypsin were purchased from Keygen Biotech Co., Ltd (Nanjing, China). All other chemicals used were of the highest commercial grade.

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Cell culture Rat pheochromocytoma PC12 cell line was provided by Keygen Biotech Co., Ltd. (Nanjing, China). Cells were cultured in DMEM supplemented with 10% heat-inactivated newborn bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO2 humidified atmosphere. For differentiation, cells were added 50 ng/mL nerve growth factor (NGF) for 8 days. When used, cells were treated with different concentrations of TCBQ for the indicated times. Control groups were treated with the same amount of DMSO. For some assays, NAC (5 mM), GSH-E (5 mM) and BSO (100 μM) were pre-incubated for 12 h prior to treatment with 20 μM TCBQ. Protein extraction and Western blotting analysis Nuclear and cytosolic proteins were prepared using Nuclear/Cytosol Fractionation Kit according to the manufacturer's protocol and our previous publication.32 Total proteins were prepared as described previously.19 Then 30-50 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (PALL). After blocked with 5% BSA, the membranes were probed with appropriate primary antibodies overnight at 4°C and further incubated with a horseradish peroxidase (HRP)-coupled secondary antibody for 1 h. Detection was performed using BeyoECL Star. GAPDH was used as a loading control. Densitometry was performed using Image J software and representative blots were chosen from at least three separate experiments showing the similar results. Immunofluorescence double staining PC12 cells grown up to 60–80% on confocal dishes and treated with 20 μM TCBQ for 6 h. Cells

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were fixed in 4% paraformaldehyde for 30 min, permeabilized in 1% Triton X-100 for 10 min, and blocked in 10% BSA for 1 h. Then cells were incubated with appropriate Nrf2 polyclonal antibody for 1 h followed by incubation with goat anti-rabbit (Alexa Fluor 488) for an additional 1 h. Finally, cells were visualized using a fluorescence microscopy (OLYMPUS IX71). CCK8 assay Cell viability after exposure to TCBQ with or without Nrf2 knockdown was determined by Cell Counting Kit-8 (CCK-8) Assay (Bimake, USA). Briefly, PC12 cells (5×103 cells /well) were seeded in 96-well culture plates. After treatment with TCBQ, the medium was replaced with fresh DMEM containing 10% kit reagent and incubated at 37°C for 2~4 h. The absorbance of the plates were measured at 450 nm using an ELx800 microplate reader (BioTek Instruments Inc., USA). LDH assay Cell viability was calculated by measuring LDH leakage from the damaged cells using LDH Kit according to the manufacturer's protocol. The activity of LDH release in the media represented the integrity of the cell membrane. PC12 cells (4×104 cells/well) were seeded in 24-well culture plates. After treatment with TCBQ, the cell culture medium was centrifuged to collect the supernatant. The amount of LDH released in the supernatant was determined according to the manufacturer's instructions. All experiments were carried out in triplicate. Annexin V-FITC/PI staining Determination of cell death was performed by flow cytometry assay using Annexin V-FITC Apoptosis Detection Kit according to manufacturer's protocol. Briefly, cells were trypsinized and washed twice with cold PBS. Then, cell pellet was resuspended with binding buffer containing 8


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fluorescein isothiocyanate (FITC)-conjugated annexin-V/propidiumiodide (PI) double dye for 20 min at room temperature in the dark. Fluorescence intensity of cells was immediately determined with a BD FACS Vantage SE flow cytometer (BD Biosciences). Data was collected from 10,000 cells per sample, and the percentage of apoptosis/necrosis cells was determined by BD Cell Quest software. Small RNA (siRNA) interference For the silencing of Nrf2 gene, PC12 cells were grown to 30%~50% confluence, and then transfected with 25 nM Nrf2-targeted siRNA or control scrambled siRNA using a siRNA-mate transfection reagent (Shanghai GenePharma Co. Ltd.) according to the manufacturer's protocol. At 48 h after transfection, cells were stimulating with or without TCBQ for the indicated times. The target sequences for Nrf2 were 5'-UUA AGA CAC UGU AAC UCG GGA AUG G-3' (forward) and 5'-CCA UUC CCG AGU UAC AGU GUC UUA A-3' (reverse). Glutathione assay PC12 cells (2.5×105 cells/well) were seeded in 6-well culture plates and grown overnight. After treatment with TCBQ, cells were collected and lysed by liquid nitrogen. The lysate was centrifuged at 4,000 g (10 min, 4°C) extracts were centrifuged to remove cellular debris, then the supernatant was assayed for intracellular glutathione by Glutathione Assay Kit according to the manufacturer's instructions. The glutathione levels were standardized by protein content as determined using a BCA assay kit (Dingguo Biotechnology Co., Ltd. China). RT-qPCR Total RNA was extracted using RNA Purification Kit (BioTeke, China) following the

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manufacturer's protocol, and was then reverse transcribed into cDNA by All-in-One cDNA Synthesis SuperMix (Bimake, USA). Expression levels of SLC7A11 and SLC3A2 mRNA were evaluated in triplicate by RT-qPCR using 2× SYBR Green qPCR Master Mix (Bimake, USA). Gene-specific primers were purchased from Sangon Biotech Co., Ltd (Shanghai, China) and listed in Table 1. Data from three independent experiments was analyzed, and the 2− △ △ Ct method was used for quantification of gene expression.33 β-actin was used as a housekeeping gene. Fluorescence labeling of protein thiol groups The depletion of protein-SH was an evidence of protein damage. Detection of the levels of protein thiols groups using dibromobimane (bBBr) assays was performed as described previously with minor modifications.34 Dibromobimane could react with reduced thiols to produce strong fluorescent adducts, then, the levels of the reduced protein-SH were examined by fluorescence intensity. Briefly, 5×105 cells treated with or without TCBQ were collected in PBS, and then perchloric acid (6 N) was added immediately to precipitate the proteins. The volume ratio of PBS and perchloric acid was 3:1. The samples were incubated on ice for 5 min and centrifuged at 14000 g for 10 min. After discarding the supernatant, the precipitated proteins were dissolved in an appropriate amount of 0.1 M NaOH and subsequently neutralized with 0.5 M Tris-HCl to PH 7.2. Dibromobimane was dissolved in DMSO into a stock concentration of 4 mM and was diluted to a final concentration of 40 μM in the protein solution. After incubated for 40 min at 37°C, the fluorescence intensities were determined by a fluorescence spectrophotometer (Hitachi, Japan) set at Ex/Em 393/477 nm. Fluorescence was normalized by total proteins and was expressed as a percentage of the control samples. Subcellular fractionation 10


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Preparation of cytosolic, mitochondrial, nuclear, and ER-microsomal cell fractions was performed as described in our previous publication.19 Briefly, 5×107 cells were trypsinized, collected, centrifuged and washed with PBS. Then, cells were resuspended in a homogenizing buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, 25 mM KCl, 1 mM EDTA and a proteinase inhibitor cocktail) and homogenized 30-50 passes with an ice-cold dounce tissue homogenizer. The homogenate was centrifuged at 1,000 g (10 min, 4°C) and the post-nuclear supernatant was centrifuged at 10,000 g (10 min, 4°C). Then the post-mitochondrial supernatant was centrifuged at 100,000 g (60 min, 4°C). This supernatant after ER-microsomal fraction was the cytosolic fraction. The subcellular fractions of induced PC12 cells were used for protein-SH measurements. Statistical analysis The Data was collected from at least three independent experiments and presented as mean ± SD. SPSS 19.0 statistical software was used for statistical analysis between different groups with one-way ANOVA followed by least significance difference (LSD) multiple comparison tests. p

Tetrachlorobenzoquinone-induced Nrf2 Confers Neuron-Like PC12

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