Article pubs.acs.org/crt
Polychlorinated Biphenyl Quinone Metabolite Promotes p53-Dependent DNA Damage Checkpoint Activation, S‑Phase Cycle Arrest and Extrinsic Apoptosis in Human Liver Hepatocellular Carcinoma HepG2 Cells Xiufang Song,† Lingrui Li,† Qiong Shi,† Hans-Joachim Lehmler,*,‡ Juanli Fu,† Chuanyang Su,† Xiaomin Xia,† Erqun Song,† and Yang Song*,† †
Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, People’s Republic of China ‡ Department of Occupational and Environmental Health, College of Public Health, The University of Iowa, Iowa City, Iowa 52242, United States ABSTRACT: Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants. The toxic behavior and mechanism of PCBs individuals and congeners have been extensively investigated. However, there is only limited information on their metabolites. Our previous studies have shown that a synthetic PCB metabolite, PCB29-pQ, causes oxidative damage with the evidence of cytotoxicity, genotoxicity, and mitochondrial-derived intrinsic apoptosis. Here, we investigate the effects of PCB29-pQ on DNA damage checkpoint activation, cell cycle arrest, and death receptor-related extrinsic apoptosis in human liver hepatocellular carcinoma HepG2 cells. Our results illustrate that PCB29-pQ increases the S-phase cell population by down-regulating cyclins A/D1/E, cyclin-dependent kinases (CDK 2/4/6), and cell division cycle 25A (CDC25A) and up-regulating p21/p27 protein expressions. PCB29-pQ also induces apoptosis via the up-regulation of Fas/FasL and the activation of caspase 8/3. Moreover, p53 plays a pivotal role in PCB29-pQ-induced cell cycle arrest and apoptosis via the activation of ATM/Chk2 and ATR/Chk1 checkpoints. Cell cycle arrest and apoptotic cell death were attenuated by the pretreatment with antioxidant N-acetyl-cysteine (NAC). Taken together, these results demonstrate that PCB29-pQ induces oxidative stress and promotes p53-dependent DNA damage checkpoint activation, S-phase cycle arrest, and extrinsic apoptosis in HepG2 cells.
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process of drinking water9,10 or the photocatalytic degradation of triclosan.11 A Fenton-like oxidation reaction proved to be highly efficient in the degradation of chlorophenols and yielded condensation byproducts, such as catechol, hydroquinone, and quinone.12 Once quinones are formed, they readily react with nucleophiles via Michael addition13 or by the chlorine substitution reaction.13 In biological systems, reactions of quinones with cellular macromolecules, such as DNA or proteins, can result in cellular dysfunction and toxicity. For example, the formation of DNA adducts by quinone metabolites of PCBs14 and PBDEs6 have been described previously. In addition, highly reactive semiquinone radical intermediates can be formed from a range of environmental pollutants during the futile redox-cycling of hydroquinone−quinone couples.15,16 This redox-cycling process can result in oxidative stress, an imbalance in the production of reactive oxygen species (ROS) (i.e., hydrogen peroxide, hydroxyl radicals, and superoxide anion radicals), and quinone-mediated toxicity.
INTRODUCTION Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, whose production has been banned including most of their uses in most industrialized countries from the 1970s. However, due to their persistent physicochemical properties, PCBs become widely distributed geographically and accumulate in plants and animals through the food chain.1 The diverse toxicities of PCBs have been extensively documented, including endocrine disruption, neurotoxicity, immunotoxicity, etc.2 Although PCBs are highly stable, they have a tendency to metabolize under various conditions.3 Many well-studied environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH)s,4 benzo[α]pyrene (B[α]P),5 and polybrominated diphenyl ethers (PBDEs),6 are readily metabolized into quinones via their corresponding hydroxylated forms. Similarly, chlorinated aromatic compounds, including chlorobenzene,7 pentachlorophenol,8 as well as PCBs, undergo bioactivation into the chlorinated quinone. In addition to biotransformation, environmental and technical processes may result in the formation of quinones. For instance, chlorinated quinones were formed as byproducts of the chlorination © 2015 American Chemical Society
Received: July 31, 2015 Published: October 9, 2015 2160
DOI: 10.1021/acs.chemrestox.5b00320 Chem. Res. Toxicol. 2015, 28, 2160−2169
Article
Chemical Research in Toxicology
Ac-DEVD-CHO, Hoechst 33258, and RIPA lysis buffer were purchased from Beyotime Institute of Biotechnology (Haimen, China). Ataxia telangiectasia mutated (ATM), ATM Rad3-related protein (ATR), Chk1, Chk2, p53, cyclin A, cell division cycle 25A (CDC25A), CDK 4, CDK 6, Fas, caspase 3, and caspase 8 antibodies were purchased from Proteintech Group Co. Ltd. (Wuhan, China). Antibodies against p-p53, p21CIP1/WAF1, cyclin D1, cyclin E, cyclindependent kinase 2 (CDK 2), and β-actin antibodies were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Antibody against Fas ligand (FasL) was purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Antibodies against p-ATM and p-ATR were obtained from Santa Cruz Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). HRP-conjugated goat antirabbit IgG (H+L) antibody, FITC-labeled goat antirabbit IgG (H+L) antibody, Alaxa Fluor 555-labeled Donkey antirabbit IgG (H+L) antibody, Triton X-100, and EDTA were supplied by Dingguo Biotechnology Co. Ltd. (Beijing, China). DMEM medium was obtained from Gibco Co. (Grand Island, NY, USA). p53 siRNA, sham siRNA, and siRNA-mate transfection reagent were purchased from GenePharma Co. Ltd. (Shanghai, China). Other chemicals were of the highest grade commercially available and used without further purification. Cell Culture and Treatment. The HepG2 cell line was purchased from Third Military Medical University, Chongqing, China. Cells were cultured in DMEM medium with 10% fetal bovine serum (HyClone, USA) and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) at 37 °C under a 5% CO2 atmosphere. MTT Assay. In brief, cells were collected and seeded in 96-well culture plates at 1 × 104 cells/well. Cells were permitted to adhere overnight at 37 °C and incubated with PCB29-pQ. MTT solution (10 μL; 5 mg/mL in PBS) was added to each well, and the plates were maintained at 37 °C for 4 h. For N-acetyl-cysteine (NAC) treatment and p53 or caspase inhibition assays, cells were treated with 5 mM NAC, 5 μM pifithrin-α (p53 inhibitor), 5 μM Z-VAD-FMK (pan caspase inhibitor), or 5 μM Ac-DEVD-CHO (caspase 3 inhibitor) for 1 h before culturing with PCB29-pQ. MTT-containing medium was aspirated off, and 100 μL of DMSO was added to each well and mixed thoroughly for 10 min using a shaker. The formazan product was measured for its optical density (OD) at 570 nm using a microplate reader (BioTek ELX800, Vermont, USA). Experiments were conducted in triplicate, and results were expressed as a percentage of DMSO-treated controls. Flow Cytometry Analysis. Flow cytometry analysis was performed to investigate PCB29-pQ-induced cell cycle arrest and the frequency of apoptotic cells. (a) Cell cycle arrest assay: After treatment with PCB29-pQ for the indicated times, HepG2 cells were collected by centrifugation at 1,000g for 5 min and washed with icecold PBS. The cell pellet was fixed with ice-cold 70% ethanol overnight at 4 °C and stained with PI for 30 min at 4 °C in the dark. Cell cycle distribution was analyzed with an Epics XL flow cytometer
Oxidative stress has been implicated in multiple cellular events, including cell cycle arrest and apoptosis.17 In order to protect the integrity of the genome from oxidative attack, a series of genes is activated to induce cell cycle arrest and initiate corresponding repair pathways. Alternatively, apoptosis is triggered through mitochondria and/or death receptormediated pathways to ensure an orderly dismantling of damaged cells when repair cannot be executed. Quinones formed from lower chlorinated PCBs represent a particular environmental concern because of their well-documented role in the genotoxicity and carcinogenicity of this ubiquitous group of persistent organic pollutants.18,19 There is considerable evidence that PCB quinones cause ROS formation.15,20 PCB quinones delay the entry of quiescent MCF-10A human breast epithelial cells into its proliferative cycle21 and, as we recently reported, induce mitochondrial-dependent apoptosis.22 However, it is currently unknown whether PCB quinones cause cell cycle arrest and induce death receptor-dependent apoptosis. The signaling pathways involved in cell cycle arrest and mitochondria (or death receptor)-dependent apoptosis are similar across different species23 and, therefore, important for understanding the toxicity of many quinones in wildlife and humans. To gain further insights into the environmental toxicity of PCB and, possibly, other environmental quinones, we analyzed the effects of 2,3,5-trichloro-6-phenyl-[1,4]benzoquinone (PCB29-pQ), a putative quinone metabolite of PCB29, on cell cycle distribution and death receptor-related apoptotic signaling pathway using the human hepatocellular liver carcinoma (HepG2) cell line. The HepG2 cell line was selected for these studies as a well-characterized model for the effects of environmental contaminants, such as halogenated biaryl ethers,24 nanoparticles,25 organic drinking water disinfection byproducts,26 and heavy metals.27 Our results with PCB29-pQ provide novel insights into the mechanisms of quinone-mediated toxicity that are likely shared by other environmental quinones.
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MATERIALS AND METHODS
Chemicals and Reagents. PCB29-pQ was synthesized and characterized as previous described28 and maintained in a −20 °C freezer under argon atmosphere. A stock solution of PCB29-pQ (10 mM) was prepared in DMSO before use. An equal amount of DMSO (
