Quinones Derived from Polychlorinated Biphenyls Induce ROS

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Polychlorinated biphenyl quinone induces ROS-dependent autophagy through the evoke of autophagic flux and mTOR/p70S6k inhibition in two independent cell lines Qiong Shi, Xiufang Song, Zixuan Liu, Yawen Wang, Yuxin Wang, Juanli Fu, Chuanyang Su, Xiaomin Xia, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00127 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 13, 2016

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Chemical Research in Toxicology

Quinones derived from polychlorinated biphenyls induce ROSdependent autophagy by evoking an autophagic flux and inhibition of mTOR/p70S6k

Qiong Shi, Xiufang Song, Zixuan Liu, Yawen Wang, Yuxin Wang, Juanli Fu, Chuanyang Su, Xiaomin Xia, 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:

Phone: +86-23-68251503. Fax: +86-23-68251225. E-mail addresses: [email protected] or [email protected]

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TABLE OF CONTENTS

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ABSTRACT Autophagy is a “self-eating” destructive process, which eliminate damaged organelles to maintain cellular homeostasis. Polychlorinated biphenyls (PCBs) are one of the most infamous industrial pollutants, which are ubiquitous in nature. In our present study, we found that an active, quinone-type PCB metabolite (PCB29-pQ) treatment causes autophagic response through mTOR/p70S6k inhibition in HepG2 and MDA-MB-231 cells. Furthermore, our data suggested that PCB29-pQ enhances autophagosome formation through autophagic vacuole (AV) biogenesis, which evokes autophagic flux and induces AV-lysosome co-localization. The inhibition of autophagy enhanced PCB29-pQ-caused cytotoxicity suggested autophagy serves as a pro-survival machinery, which played a protective role in the early stage of PCB29-pQ-induced insult. However, higher concentration of PCB29-pQ exposure (>5 µM) caused autophagic cell death, which implied the shift of “pro-survival” to “pro-death” upon autophagic signaling. NAcetylcysteine (NAC) suppressed PCB29-pQ-induced autophagy and cytotoxicity suggested that ROS plays an important role in the regulation of PCB29-pQ-induced autophagy. Since autophagy shows significant implications in various human diseases and conditions, our current study provide a new mechanism of PCB-associated toxicities.

Keywords: PCB; quinone; autophagy; mTOR; LC3B; ATG

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INTRODUCTION Polychlorinated biphenyls (PCBs) are a large group of persistent industrial contaminants comprising 209 congeners, which are widely distributed in the environment.1 Although PCBs production was banned in 1970’s, PCBs were still found in various environmental matrices due to several reasons. The first reason is that PCBs are strongly resistant to degradation in the environment; the second reason is PCBs may leak from the older devices for their inappropriate disposal; the third reason is PCBs may currently be produced as unintentional byproducts in the manufacture of paints and pigments.2 PCBs exposure are associated with immunotoxicity, neurotoxicity, hepatotoxicity, lipotoxicity and potential cancer risks to humans.1, 3 In fact, PCBs have been promoted to a group 1 human carcinogen by International Agency for Research on Cancer (IARC).4 Our previous studies illustrated a highly reactive PCB metabolite, PCB29-pQ exposure cause reactive oxygen species (ROS) generation and then bring various detrimental effect, including viability loss, DNA damage, mitochondrial destruction, endoplasmic reticulum (ER) stress and apoptosis in vitro and in vivo.5-9 Like other stresses, oxidative stress can result in various responses. For example, mild oxidative stress-induced DNA damage is frequently linked with the activation of transcription factors and cell cycle arrest to allow repair of DNA damage. Indeed, our recent study demonstrated that PCB29-pQ promotes p53-dependent DNA damage checkpoint activation, S-phase cell cycle arrest and DNA repair signaling in HepG2 cells.5, 7 Other adaptive responses may apply and need further investigation. Autophagy is an intracellular degradation process for the turnover of cellular organelles in response to different stimuli.10 In general, there are several steps in the process of autophagy. Initially, the cytoplasmic materials marked for degradation, including unwanted/damaged 4


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organelles, ubiquitinated substrates or aggregates protein, were sequestered into a vesicular membrane phagophore. The elongation/maturation of phagophore leads to the formation of a double-membrane vesicle autophagosomes. Then, it fuse with lysosomal membrane thereby form autolysosomes, and is finally degraded by lysosomal proteases. Autophagy has “pro-survival” function by degrading impaired organelles or aggregated proteins in maintaining cellular homeostasis and preventing harmful consequences.11 On the contrary, autophagy can also be “pro-death” under certain situations, which was termed as autophagic cell death.12 Oxidative stress stimulation was identified to induce autophagy in different cellular systems. However, their exact molecular mechanisms remains controversial. Thus, it is very important to decipher the mechanisms that underlie the divergence between adaptive responses upon oxidative stress. Our previous study showed that PCB29-pQ induces oxidative damage in a doseand time-dependent manner. Thus, the authors further hypothesized that PCB29-pQ induces autophagy via ROS-dependent mechanism.

MATERIALS AND METHODS Materials and Reagents. 2, 3, 5-trichloro-6-phenyl-[1, 4]-benzoquinone (PCB29-pQ, the structure is shown in Figure 1A) was synthesized and characterized as previously described.13 Acridine orange (AO) was purchased from Amresco (Solon, OH, USA). Rapamycin (Ra) was purchased from YuanYe Bio Technology Co, Ltd. (Shanghai, China). Chloroquine diphosphate (CQ) was supplied by Heowns Biochem LLC (Tianjin, China). 3-Methyladenine (3-MA) was obtained from Aike Reagent (Chengdu, China). Cell counting kit-8 (CCK-8) kits was obtained from Genview (Shanghai, China). 4’, 6-Diamidino-2-phenylindole dihydrochloride (DAPI) and Lyso-Tracker Red were supplied by Beyotime Institute of Biotechnology (Nanjing, China). 5



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Rhodamine (TRITC)-conjugated AffiniPure goat anti-rabbit IgG(H+L) was purchased from ZSGB-BIO (Beijing, China). Monodansylcadaverine (MDC) and rabbit light chain 3B (LC3B) polyclonal primary antibody were purchased from Sigma-Aldrich Inc. (Shanghai, China). Rabbit autophagy specific genes 5 (ATG5), ATG12, p62, lysosomal-associated membrane protein 2 (LAMP2), cleaved-caspase3, mouse LC3B and β-actin polyclonal primary antibodies, Alexa Fluor 488-labeled goat anti-mouse IgG(H+L) were obtained from Proteintech group, Inc. (Wuhan, China). Mammalian target of rapamycin (mTOR), phosphorylated mTOR, p70 ribosomal S6 kinase (p70S6K) and phosphorylated p70S6K polyclonal primary antibodies were purchased from Ruiying Biological (Suzhou, China). Goat-anti rabbit IgG-HRP conjugated secondary antibody was supplied by Sangon Biotech Co., Ltd. (Shanghai, China). GFP-LC3 plasmid was a kind gift provided by Prof. Canhua Huang, Sichuan University. All other chemicals used were of the highest commercial grade. Cell Culture. Human hepatoma HepG2 and MDA-MB-231 cells were purchased from Nanjing KeyGEN Biotech. Co. Ltd. (Nanjing, China). Cells were cultured in DMEM containing 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co. Ltd.), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37°C and 5% CO2 incubator. PCB29-pQ was treated for indicated time and concentration. HepG2 and MDA-MB-231 cells were simultaneous exposed with CQ (or 3-MA) and PCB29-pQ. As for antioxidant treatment, cells were pretreated with 5 mM NAC for 1 h, then incubated with 5 µM PCB29-pQ for 24 h. Cell Viability Assay. Cell viability was assessed by CCK-8 assay. Briefly, cells were seeded into 96-well plates at 5×103 cells/well contain 100 µL medium and cultivated for 12 h to adhere. After treatment with 0 to 40 µM of PCB29-pQ for indicated time, 10 µL of kit reagent was added

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to cells followed with 3 h incubation. The OD value was read at 450 nm using a microplate reader (BioTek ELX800). Transmission Electron Microscopy (TEM). Cells were plated onto dish (60 mm) at 1×106 cells/ml with 5 mL medium and cultivated for 24 h to adhere. To observe the microstructure of HepG2 and MDA-MB-231 cells, TEM analysis was performed. After being treated with 5 µM PCB29-pQ for 24 h, HepG2 and MDA-MB-231 cells were harvested with 0.1% trypsin-EDTA solution and washed twice with PBS. Afterwards, cells were fixed with cold glutaraldehyde for 3 h. Then, cells were postfixed with 2% osmium tetroxide and embedded in Epon. Then, 0.1 mm thin sections were stained with uranyl acetate/lead citrate and visualized in a Hitachi-7500 TEM instrument (Hitachi, Tokyo, Japan). Detection of Acidic Vesicular Organelles (AVOs). The formation of AVOs (a morphological characteristic of autophagy) was detected by AO staining. HepG2 and MDA-MB231 cells were seeded into 6-well plates at 3×105 cells/ml with 2 mL medium and cultivated for 24 h to adhere. After treatment with 0 to 20 µM of PCB29-pQ for 24 h, then cells were stained with 1 µg/ml AO at 37°C for 20 min, cells were washed twice with PBS and were observed immediately under a fluorescence microscope (Olympus IX71). Visualization of Autophagic Vacuoles (AVs). Here, MDC was introduced as a specific autolysosomal marker for autophagic process analysis. HepG2 and MDA-MB-231 cells were seeded into 6-well plates at 3×105 cells/ml with 2 mL medium and cultivated for 24 h to adhere. Cells were treated with 5 µM PCB29-pQ for 24 h, the autophagic vacuoles were stained with 50 µM MDC at 37°C for 30 min. After incubation, the cells were washed twice with PBS and immediately viewed using a fluorescence microscope (Olympus IX71). The MDC fluorescence

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intensity analysis was performed by Image-pro plus software and was expressed as relative MDC fluorescence compare to untreated control cells. Protein Preparation and Quantification. HepG2 and MDA-MB-231 cells were plated onto dish (60 mm) at 1×106 cells/ml with 5 mL medium and cultivated for 24 h to adhere. After being treated with PCB29-pQ, HepG2 and MDA-MB-231 cells were washed twice with PBS and lysed with RIPA lysis buffer. The cellular lysates were centrifuged at 10,000 g and 4°C for 10 min. The supernatant proteins were collected and stored at -20°C until used. Western Blotting. The whole-cell lysates were separated by 8% or 12% SDS-PAGE and transferred onto the nitrocellulose membranes. The membranes were blocked with 5% BSA or 5% nonfat dry milk at 37°C for 2 h. Then, the membranes were incubated with primary antibodies at 4°C overnight and appropriate secondary antibodies conjugated to horseradish peroxidase at room temperature for 2 h. The proteins were detected using the ECL system or the HRP substrate DAB system. β-Actin was used as loading controls. Densitometric analysis of immunoblot was performed by ImageJ software. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR). HepG2 and MDA-MB231 cells were plated onto dish (60 mm) at 1×106 cells/ml with 5 mL medium and cultivated for 24 h. After treating HepG2 and MDA-MB-231 cells with PCB29-pQ, total RNA was extracted with a total RNA purification kit (BioTeke, Beijing, China) as described in the manufacturer’s manual. RNA was reversely transcribed into cDNA which was used to perform quantitative realtime PCR analysis using LightCycler 96 instrument protocol with Faststart Essential DNA Green Master (Roche, Switzerland). The oligonucleotide primers, forward, 5’-ATG TCA ACA TGA GCG AGT TGG T-3’, and reverse, 5’-CTG GTT CAC CAG CAG GAA GAA-3’, were used to amplify human LC3 in HepG2 cells. The primers forward, 5’-GAT GTC CGA CTT ATT CGA 8


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GAG C-3’ and reverse, 5’-TTG AGC TGT AAG CGC CTT CTA-3’, were used to amplify human LC3 in MDA-MB-231 cells. The primers forward, 5’-TCC TCC CTG GAG AAG AGC TAC-3’, and reverse, 5’-TCC TGC TTG CTG ATC CAC AT-3’, were used to amplify β-actin. Three independent replicates were analyzed per sample, and relative gene expression normalized to the internal housekeeping gene β-actin was obtained by the 2−∆∆Ct method. LC3B Turnover Assay. The LC3B turnover assay measured the amount of LC3B-II delivered to the lysosomes by comparing the LC3B-II amounts in the presence or absence of lysosome inhibitor CQ (final concentration of 10 µM). HepG2 and MDA-MB-231 cells were seeded at cell densities (1×106 cells) in culture dish (60 mm) with 5 mL medium and allowed to adhere and grow for 24 h before exposure to PCB29-pQ. Cell Transfections and Microscopy. Cells were plated onto confocal dish (35 mm) at 1.5×105 cells/ml with 2 mL medium and cultured for 12 h to adhere. Then cells were transfected with GFP-LC3 plasmid using the RNAi-Mate (GenePharma, Shanghai, China) according to the manufacturer’s instructions. 1.6 µg of GFP-LC3 plasmid and 5 µL of RNAi-Mate were mixed together with Opti-MEM and incubated at room temperature for 20 min to allow the formation of complexes. Then, cells were incubated in 10% serum medium for 24 h followed PCB29-pQ exposure for additional 24 h. Cell lysosomes and nuclei were stained using Lyso-Tracker Red and DAPI respectively. For AVs and LAMP2 staining, cells were incubated with mouse antiLC3B and rabbit anti-LAMP2 respectively, followed by the incubation of goat anti-mouse (Alexa Fluor 488) and goat anti-rabbit secondary antibody (TRITC). Finally, cells were analyzed by a fluorescence microscope (Olympus IX71). Flow Cytometry Analysis for Apoptosis. The frequency of apoptotic cells was detected by flow cytometry assay. HepG2 and MDA-MB-231 cells were seeded at cell densities (1×106 cells) 9



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in culture dish (60 mm) with 5 mL medium and allowed to adhere and grow for 24 h before exposure to PCB29-pQ. After treatment with PCB29-pQ for 24 h, cells were washed twice with cold PBS, then re-suspended with binding buffer and incubated with FITC-conjugated annexinV/propidium iodide (PI) double staining solution for 15 min according to manufacturer’s instructions. Fluorescence was determined on a BD FACS Vantage SE flow cytometer (BD Biosciences) and the percentage of apoptotic cells was calculated using BD Cell Quest software. Statistical Analysis. All data were expressed as the mean ± standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by least significance difference (LSD) multiple comparison tests using SPSS19 software. Differences were considered statistically significant at P

Quinones Derived from Polychlorinated Biphenyls Induce ROS

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