Polychlorinated biphenyl quinones promotes ... - ACS Publications

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Polychlorinated biphenyl quinones promotes breast cancer metastasis through ROS-mediated nuclear factor kappa B-matrix metalloproteinase signaling Yuxin Wang, Yawen Wang, Zixuan Liu, Wenjing Dong, Bingwei Yang, Xiaomin Xia, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00148 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Chemical Research in Toxicology

Polychlorinated biphenyl quinones promotes breast cancer metastasis through ROS-mediated nuclear factor kappa B-matrix metalloproteinase signaling

Yuxin Wang, Yawen Wang, Zixuan Liu, Wenjing Dong, Bingwei Yang, 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: College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing, People’s Republic of China, 400715. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail addresses: [email protected] or [email protected]

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Table of Content

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ABSTRACT

It is generally acknowledged that polychlorinated biphenyls (PCBs) exposure increased the incidence of cancer, however, the underlying mechanism(s) of PCBs-induced cancer metastasis are unclear. PCBs is readily to metabolize, little information is available regarding the effect of PCBs metabolites on cancer metastasis. Currently, we evaluated a high reactive PCBs metabolite, 2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone (PCB29-pQ), relevant exposure with mammary cancer metastasis. Multiple line of evidences illustrated that PCB29-pQ induces breast cancer invasion and migration. In particular, this appearance is associated with two-fold elevation of matrix metalloproteinases-2/-9 (MMP-2/-9) and extracellular nuclear factor kappa B (NF-κB), respectively. Our results clearly demonstrated the translocation of cytosolic NF-κB into the nucleus, by a factor of about 2.4. We also revealed the activation of corresponding upstream signaling cascades phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and p38, and extracellular regulated protein kinases (ERK) mitogen-activated protein kinase (MAPK), by factors of 3.15, 3.09 and 1.69, respectively. Moreover, there was a marked induction of reactive oxygen species (ROS) after PCB29-pQ challenge and antioxidant treatment markedly inhibited PCB29-pQ-mediated activation of these axis signaling. Collectively, our result suggested that PCB29-pQ induces breast cancer metastasis via ROS-dependent NF-κB-MMP signaling.

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INTRODUCTION

Polychlorinated biphenyls (PCBs), a group of synthetic aromatic organochlorine compounds, were extensively used in different commercial and industrial applications, including electrical transformers, plasticizers and capacitors. Although the production of PCBs was banned in the 1970’s, unfortunately, due to their stable chemical-physical properties, there are more than 1.5 million tons of PCBs still existed in the environment and spread globally.1 PCBs may enter organism through food chain, respiratory and dermal exposure, and accumulate in tissues. The adverse effects of PCBs have been well documented in numerous animal and human studies, including reproductive, immunologic, genomic and endocrine adverse effects.2 Breast cancer is one of the most commonly malignant tumor with serious health challenge. It was estimated 268,670 new cases of breast cancer diagnosed and 41,400 breast cancer deaths in the United States in 2018.3 Metastasis are the major reason of cancer-related poor clinical treatment and high mortality, therefore, it is important to unveil underlying mechanisms that regulate metastasis. The development of metastasis involves the detachment of cancer cells from primary tumor, then, transferred to lung, liver and other organs. Recent advancements in etiology have discovered several complex and diverse signaling pathways implicated in breast cancer metastasis.4 Although PCBs was promoted to Group 1 carcinogenic to humans by the International Agency for Research on Cancer in 2016,5 currently, the evidence for an association between total PCBs and breast cancer risk was inconsistent.6 An early case-control study reported the positive association in the occurrence of breast cancer among women with high serum level of PCBs,7 however, the major follow-up studies do not provide robust support for a role of PCBs in breast cancer.8, 9 Even though, a great number of mechanismic studies were carried out. PCBs, at least part of congeners, have 4


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structures similar to endogenous hormones, which willing to interact with hormone transport proteins or disrupt hormone metabolic pathways. A substantial number of studies illustrated the ability of PCBs on the disruption of endocrine function, that tight up with the etiology of estrogen-sensitive breast cancer. Other recent studies have focused on the contribution of genetic polymorphisms on the association between PCB and breast cancer risk.10 A higher breast cancer risk linked with high PCB exposure with CYP1A1 genetic variant was found, since cytochrome P4501A1 is readily induced by PCBs.11 Hoyer et al reported that high PCB exposure was associated with increased breast cancer risk among those women carry variants p53 suppressor gene.12 Nevertheless, a study found negative correlation between putative high-risk polymorphisms, including GSTM1, GSTT1, GSTP1 and COMT towards serum PCBs level and breast cancer.13 The primary interested outcome was PCBs-induced incident breast cancer, only few studies examined the effect of PCBs on breast cancer development.14-16 Thereafter, the lack of effective evidence impede the comprehensively understanding of complex network of signaling pathways that drives the multistep process of the metastatic cascade. Our previous studies demonstrated that PCBs have a tendency to biotransformation, resulting in high reactive quinone-type PCB metabolites and a substantial downstream reactive oxygen species (ROS).17 Interestingly, ROS plays a vital role in the initiation and progression of breast cancer, through the affection of growth factors and mitogenic pathways, and controls cellular processes. The role of ROS in the occurrence and progression of breast cancer have been extensively reviewed.18-20 In this study, we aim to investigate the effect of PCB29-pQ on breast cancer metastasis and unveil its underlying molecular mechanisms.

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MATERIALS AND METHODS Chemicals and reagents

2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone 2-(4-chlorophenyl)-[1,4]-benzoquinone

(PCB29-pQ), (PCB3-pQ)

and

2-chloro-5-(4-chlorophenyl)-[1,4]-benzoquinone (PCB15-pQ) were synthesized as previously described.21 4-chlorobiphenyl (PCB3) and 4,4’-dichlorobiphenyl (PCB15) were synthesized as previously described.22 Compounds were dissolved in dimethyl sulfoxide (DMSO) (cell culture) or corn oil (animal study) before use. The antibodies against c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases (ERK) 1/2, p38 by Wanleibio Co., Ltd. (Shenyang, China). The antibodies against p-JNK, p-ERK1/2, p-p38, p-Akt (Ser473), and p-p65 were supplied from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). The antibody against β-actin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). The antibodies against matrix metalloproteinases (MMP)-2, MMP-9, membrane type 1 (MT1)-MMP, p65, Akt, Lamin B were obtained from Proteintech group, Inc. (Wuhan, China). 6-Diamidino-2-phenylindole dihydrochloride (DAPI), EasyBlot ECL kit, pyrrolidinedithiocarbamic acid (PDTC, NF-κB inhibitor), LY294002 [phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor], PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) were obtained from Beyotime Institute of Biotechnology (Nanjing, China). All other chemicals used were of the highest commercial grade.

Cell Culture

Human breast cancer cell MDA-MB-231 was purchased from Nanjing KeyGEN Biotech. Co. Ltd. Murine breast cancer epithelial cell line stably expressing firefly luciferase (4T1-luc) was a kind 6


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gift from Dr. Wenjuan Sun of Chongqing Medical University. Three-dimensional (3D) MDA-MB-231 and 4T1-luc were cultured using plates coated with Type I Collagen (BD Biosciences) and all cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cell lines were cultured at 37°C and 5% CO2 incubator.

Animals

Female nude BALB/c mice (4 weeks old) were purchased from Laboratory Animal Center of Chongqing Medical University. Mice were treated in standard housing condition (12 h light/dark cycle at 22°C) with free access to food and water, in accordance with the guideline of the Animal Care Committee of Southwest University. Mice were randomly divided into two groups (Control and PCB29-pQ-treated group) with at least 5 mice in each group, acclimatized for at least one week prior to use. Mice received a daily dose of PCB29-pQ (2 mg/kg body mass via gastric perfusion) for 2 weeks. The control group received equal volume of corn oil throughout. Then, mice were administered with 4T1-luc tumor cells by tail vein injection. In a parallel study, MDA-MB-231 tumor cells were introduced via orthotopic injection. Tumor growth in nude mice was observed by a bioluminescence imaging Fusion system (Vilber Lourmat, France) every 5 days. Mice which contained 4T1-luc cells group were intraperitoneal injected with 100 mg/kg D-Luciferin 10 min before bioluminescence imaging acquisition. On the 15th day of monitor, all animals were sacrificed with a procedure recommend by the Panel on Euthanasia of the American Veterinary Medical Association. Lungs, livers, kidneys and spleen were removed for imaging.

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Cell Viability Assay

Cell viability was evaluated by CCK-8 kit (Bimake, USA). MDA-MB-231 and 4T1-luc cells were seeded in 96-well plates at a density of 5 × 103 cells/well before treated with various concentrations of PCB29-pQ (1, 2 or 4 µM). After 24 hours of incubation, medium was replaced with 100 µL of fresh DMEM and 10 µL of kit reagent for 30 minutes incubation. The optical density (OD) was measured at 450 nm using a microplate reader (BioTek ELX800). Cell viability was expressed as a percentage compared with the control (untreated) cells.

Wound Healing Motility Assay

MDA-MB-231 and 4T1-luc cells were grown to confluence on a 30 mm culture dishes at 2 × 105 cells/well. Scratch wounds were created using a p10 micropipette tip. After washed three times with PBS, cells were treated with PCB29-pQ (1, 2 or 4 µM). After 24 hours of incubation, bright field images of wound healing were captured by fluorescence microscopy (OLYMPUS IX71).

Invasion and Migration Assay

Transwell chambers with 8 µm pore size (24-well plates, Corning) was coated with matrigel (BD Biosciences) and kept overnight at 37°C to form a semisolid matrix. MDA-MB-231 and 4T1-luc cells were pretreated with PCB29-pQ (1, 2 or 4 µM) for 24 hours. Cells at 1 × 105 cells/well were loaded into the upper chamber with 200 µL serum-free medium and the lower chambers with medium containing 10% FBS. After 24 h of incubation, non-migrated cells were removed by cotton swab from the upper side membrane. Cells on the lower side membrane were fixed with methanol-acetic acid (3:1) and stained with crystal violet (0.05%) at room temperature. In migration 8


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assay, MDA-MB-231 and 4T1-luc cells were seeded into a Boyden chamber does not coating matrigel. Randomly chosen fields were measured using fluorescence microscopy (OLYMPUS IX71). Immunohistochemistry

Lung and liver tissues were deparaffnized and rehydrated, incubated in 3% H2O2 for 20 min. Tissues were bathed in 97°C water for 15 min to retrieve the antigen, then incubated with 5% normal goat serum in PBS at 37°C for 30 min to block nonspecific binding. The samples were probed with respective primary antibody at dilution of 1:300 at 37°C for 2 h and then an appropriate secondary antibody. After the preceding step, slides were washed three times with TBST for 5 min. The photographs were visualized with 3, 3’-diaminobenzidine tetrahydrochloride (DAB).

Gelatin zymography assay

Conditioned media was collected after 24 h PCB29-pQ treatment. Samples were separated in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) contained 0.1% gelatin. After electrophoresis, gels were washed twice with washing buffer containing 2.5% Triton X-100. Then, gels were incubated with reaction buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM CaCl2, 1 µM ZnCl2 and 0.02% Brij 35 with shaking at 37°C for 32 h. Finally, gels were stained with 0.25% (w/v) Coomassie brilliant blue in 30% methanol and 10% acetic acid for 3 hours and destained. Proteolysis was identified as nonstaining zone in dark blue fields.

Western Blotting Analysis

Cells were lysed in ice cold RIPA buffer (Dingguo Biotech Co. Ltd., Beijing, China) supplemented with 1% protein inhibitor cocktail and 1 mM PMSF. The nuclear and cytoplasmic 9



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proteins were extracted with a nuclear-cytosol extraction kit following the manufacturer’s instructions (Sangon Biotech Co., Ltd., Shanghai, China). Proteins were analyzed by electrophoresis in 10% or 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% BSA and then incubated with primary antibody. After washed with TBST three times, the membranes further incubated with secondary goat anti-rabbit IgG-HRP-conjugated antibody. The membranes were visualized with electrochemiluminescence (ECL) substrate system. Each experiment was conducted for at least three independent times. The concentrations of different cell substrates were standardized by β-actin or Lamin B.

Immunofluorescence Staining

Cells were plated onto confocal dishes and exposed to PCB29-pQ for 24 h. After sections were washed with PBS, they were fixed in 4% paraformaldehyde including 3% sucrose for 30 min and blocked with 10% BSA for 1 h. Cells were incubated with primary antibody overnight at 4°C. The slides were washed with PBS three times, developed with Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) antibody for an additional hour. After three washes, the sections were counterstained with DAPI for 10 min. Finally, images were captured by a fluorescence microscopy (OLYMPUS IX71).

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extract was prepared from MDA-MB-231 cells to detect the DNA binding activity of NF-κB. NF-κB oligonucleotide probe (5’-AGT TGA GGG GAC TTT CCCAGG C-3’, 3’-TCA ACT CCC CTG AAA GGG TCC G-5’) was obtained from Biyotime Biotech, Co. Ltd (Shanghai, China). Briefly, nuclear extracts (10 µg of protein) were incubated with 2 µL biotin-labeled oligonucleotide 10


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probes for NF-κB (p65) in a reaction mixture containing 5.5 µL ddH2O, 2.5 µL EMSA/Gel-shift binding buffer (5×) for 20 min. Then, samples were separated on 6.5% acrylamide gels and transferred onto a Magmaprobe nylon membrane (Dingguo, Beijing, China). Membranes were incubated with blocking buffer for 30 min and dipped in blocking buffer containing streptavidin-HRP conjugate for 20 min. After washed, bands were visualized with an ECL system.

Statistical Analysis

All data were analyzed using SPSS 19.0 software and were presented as the mean ± SD from three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by least significance difference (LSD) multiple comparison test. P value of

Polychlorinated biphenyl quinones promotes ... - ACS Publications

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