2,2′,4,4′-Tetrabromodiphenyl ether promotes human neuroblastoma SH-SY5Y cells migration via the GPER/PI3K/Akt signal pathway

Abstract

Neuroblastoma is the predominant tumor of early childhood. 2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) has the highest concentration among all polybrominated diphenyl ether (PBDE) congeners in human body, particularly for children. Considering that accumulating evidences showed developmental neurotoxicity of PBDE, there is an urgent need to investigate the effects of BDE-47 on the development of neuroblastoma. This study revealed that BDE-47 had limited effects on the cytotoxicity while significantly increased the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells. This was further confirmed by the results that BDE-47 treatment significantly downregulated the expression of E-cadherin and zona occludin-1 and upregulated the expression of matrix metalloproteinase-9 (MMP-9). Silencing of MMP-9 by specific small interfering RNA significantly abolished the BDE-47-induced migration and invasion of SH-SY5Y cells. Further, the signals G protein-coupled estrogen receptor 1 (GPER)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) mediated the BDE-47-induced upregulation of MMP-9 and in vitro migration of SH-SY5Y cells since G15 (GPER inhibitor) and LY 294002 (PI3K/Akt inhibitor) significantly abolished the effects of BDE-47. Our results revealed that BDE-47 significantly triggered the metastasis of human neuroblastoma SH-SY5Y cells via upregulation of MMP-9 by the GPER/PI3K/Akt signal pathway. This study revealed for the first time that BDE-47 can promote the migration of SH-SY5Y cells. It also provided a better understanding about the metastasis of human neuroblastoma induced by environmental endocrine disruptors.

Keywords

BDE-47 MMP-9 SH-SY5Y migration invasion

Introduction

Polybrominated diphenyl ethers (PBDEs) are flame retardants commonly used in the manufacture of furniture, infant products, and electronics.¹ Recent studies revealed that they are ubiquitous in environmental samples such as indoor dust and food products.^2,3 Due to physicochemical properties, such as semivolatile, not chemically bound to substrates, and highly resistant to chemical and biological degradation, PBDEs enter the food chain, accumulate in human body, and then threaten human health.⁴ Furthermore, it was found that PBDE levels in infants and children were two- to threefold higher than that in adults.^5,6 It might be due to dust that contains very high PBDE concentrations,^7,8 which is a major exposure pathway for many chemicals in children.⁶

PBDEs are endocrine-disrupting compounds with half-lives in humans ranging from 2 to 12 years.⁹ Studies suggested that PBDE exposures are associated with altered thyroid hormone levels in pregnant women¹⁰ and infants.¹¹ More seriously, numerous studies revealed the possible neurotoxic effects of in utero and early childhood exposure to PBDEs.^12
–14 This was particularly true for 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), which has the highest concentration among all PBDE congeners in human body.^15,16 It was reported that 4-year-old Spanish children with detectable blood concentrations of BDE-47 were significantly more likely to demonstrate symptoms that need attention.¹⁷ A positive association between breast milk levels of BDEs-47 and externalizing behaviors, specifically activity/impulsivity behaviors, has been found in 220 30-month-olds.¹⁴ Accumulating evidences show that PBDE exposure in animals causes developmental neurotoxicity at doses similar to those reported in human body.¹⁸ Thus, major public health concerns surround exposure to BDE-47 for children and its potential to cause adverse neurotoxicity effects.

Neuroblastoma is the predominant tumor of early childhood, which is responsible for 7% of malignancies in patients younger than 15 years of age and represents approximately 15% of deaths from childhood cancer.¹⁹ Due to the development of metastatic dissemination, neuroblastoma has an extremely poor prognosis with the 5-year survival rate of high-risk patients as low as 20–25%.^20,21 Recent studies suggested that PBDE-47 inhibited cell viability, increased lactate dehydrogenase leakage, and induced cell apoptosis in human neuroblastoma cells in vitro.²² Further, low concentrations of BDE-47 and BDE-99 can induce synergistic oxidative stress-mediated neurotoxicity in human neuroblastoma cells.²³ Interaction of BDE-47 and PCB-153 in enhancing toxicity has been observed in neuroblastoma SH-SY5Y cells. Therefore, we hypothesized that BDE-47 can modulate the biological effects of neuroblastoma cells and then promote the development of neuroblastoma in children.

On the basis of these observations, the effects of BDE-47 on the human neuroblastoma SH-SY5Y cells were investigated. We found that BDE-47 can promote the in vitro migration and invasion of SH-SY5Y cells via the G protein-coupled estrogen receptor 1 (GPER)/protein kinase B (Akt) signal pathway. To our knowledge, this is the first study to illustrate effects of BDE-47 on migration of neuroblastoma cells.

Materials and methods

Reagents

All chemicals were of reagent grade or better and were purchased from Sigma Chemical Co. (St Louis, Missouri, USA) unless otherwise noted. BDE-47 (100% pure) purchased from AccuStandard (New Haven, Connecticut, USA) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10-mM stock solution and stored at −20°C. Primary antibodies against p-Akt (Ser⁴⁷³), Akt, E-cadherin (E-Cad), (ZO-1), N-cadherin (N-Cad), fibronectin (FN), vimentin (Vim), matrix metalloproteinase-9 (MMP-9), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Horseradish peroxidase-conjugated secondary antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA). All compounds were solubilized in DMSO. The final concentration of DMSO in the cell cultures was less than 0.5%. PrimeScript^® RT reagent kit and SYBR^® Premix Ex Taq™ were products of TaKaRa Co. Ltd (Japan). E.Z.N.A^® HP total RNA kit was bought from Omega Bio-Tek (Doraville, Georgia, USA). Medium containing 0.5% DMSO was used as the control.

Cell line and cell culture

The human neuroblastoma cell line SH-SY5Y conserved in our own laboratory were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml of penicillin, and 100 μg of streptomycin. The cells were incubated at 37°C in a 5% carbon dioxide (CO₂) atmosphere. Twenty-four hours before the experiments, the medium was removed and replaced with DMEM without phenol red, supplemented with 5% dextran-coated, charcoal-treated FBS to exclude estrogenic effects caused by the medium. Then, the cells were plated in the same medium and allowed to attach overnight. All SH-SY5Y cells used in this study were used at a low passage number (<15).

Cell viability assays

Cell viability was measured using cell counting kit-8 (CCK-8 kit) according to the manufacturer’s instruction. Briefly, SH-SY5Y cells were seeded in 96-well plates at a cell density of 1 × 10⁴ per well. After treating with BDE-47 for the indicated times, 10 μl of CCK-8 solution was added to each well for an additional 2-h period. Then, the absorbance of CCK-8 was measured at 450 nm using a microplate reader. Control cells treated with DMEM were taken as 100% viability. The experiments were repeated six times.

Wound-healing assay

For the in vitro wound-healing assay, confluent monolayers of SH-SY5Y cells were scratched using sterile pipette tips. Then, the cells were cultured in serum-free media for the indicated times. The migration distance of the cells into the scratched area was measured in five randomly chosen fields. A Zeiss LSM 510 microscope (Germany) was used to obtain images. Scale bars were generated and inserted by Linux Software Map software (LSM template version 4).

In vitro migration and invasion assay

Cell migration and invasion assays were conducted as described²⁴ with a slight modification. Briefly, the cells were serum-starved overnight. The trans wells were coated with Matrigel Matrix (BD Biosciences, Franklin Lakes, New Jersey, USA) at 20 mg/ml used for invasion assay and uncoated filters were used for migration assay. The top chamber of the trans well was loaded with 0.2 ml of 4 × 10⁵ cells/ml in serum-free media, and the bottom chamber was loaded with 0.6 ml of DMEM medium containing 0.2% FBS. The cells were treated with BDE-47 in the trans wells at 37°C in a 5% CO₂ atmosphere for the indicated times. Migrated or invaded cells were fixed, stained, and counted using phase-contrast microscopy. Each migration and invasion assay was repeated in three independent experiments.

Real-time quantitative RT-PCR analysis

After exposure to BDE-47 for 24 h, total cellular RNA of SH-SY5Y cells was extracted with TRIZOL reagent according to the manufacturer’s instructions. Total RNA of 2.0 μg was reverse transcribed to complementary DNA using oligo-dT primer and Superscript II Reverse Transcriptase (Gibco BRL, Grand Island, New York, USA). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used with Platinum SYBR Green quantitative PCR (qPCR) Super Mix-UDG and detected with ABI PRISM 7900HT sequence detection system (Applied Biosystems, Waltham, Massachusetts, USA). GAPDH was used as an internal control in parallel for each run. Primer pairs were as follows: GAPDH, forward: 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse: 5′-TGG TGA AGA CGC CAG TGG A-3′; E-Cad, forward: 5′-TAC ACT GCC CAG GAG CCA GA-3′ and reverse: 5′-TGG CAC CAG TGT CCG GAT TA-3′; ZO-1, forward: 5′-GTG TTG TGG ATA CCT TGT-3′ and reverse: 5′-GAT GAT GCC TCG TTC TAC-3′; MMP-9, forward: 5′-GCA CGA CGT CTT CCA GTA CC-3′ and reverse: 5′-CAG GAT GTC ATA GGT CAC GTA GC-3′; FN, forward: 5′-TTA TGA CGA CGG GAA GAC CT-3′ and reverse: 5′-GCT GGA TGG AAA GAT TAC TC-3′; N-Cad, forward: 5′-GGT GGA GGA GAA GAA GAC CAG-3′ and reverse: 5′-GGC ATC AGG CTC CAC AGT-3′; and Vim, forward: 5′-TGA GTA CCG GAG ACA GGT GCA G-3′ and reverse: 5′-TAG CAG CTT CAA CGG CAA AGT TC-3′. The cycle number when the fluorescence first reached a preset threshold (threshold cycle (C _t)) was used to quantify the initial concentration of individual templates for the expression of messenger RNA (mRNA) of genes of interest. All experiments were performed three times independently, and the average was used for comparison.

Western blot analysis

After treating with or without BDE-47 for the indicated times, the protein extracts from SH-SY5Y cells were mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and then denatured at 95°C for 5 min. Then, the cell lysates (20 μg total protein) of each group were loaded into lanes with 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Pall Inc., East Hills, New York, USA). Blots were blocked with 5% nonfat milk overnight at 4°C and incubated for 4 h at room temperature with polyclonal primary antibody. After washing three times with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at 1:1000 dilution for 2 h at room temperature. After being washed three times, the membranes were stained with enhanced chemiluminescence solution (Pierce Biotechnology Inc., Rockford, Illinois, USA) for 2 min. Immune-reactive bands were scanned on a laser densitometer (SynGene, Cambridge, UK).

RNA interference

SY-HY5Y cells were plated on six-well plates (2 × 10⁵ cells per well) and cultured for 24 h. They were then transfected with 100 pmol small interfering RNA (si-RNA) oligomer mixed with lipofectamine 2000 reagent in serum-reduced medium according to the manufacturer’s instructions. The target sequences for MMP-9 si-RNAs was 5′-AAC ATC ACC TAT TGG ATC CAA-3′. Both siRNA for Snail (si-Snail) and control si-RNAs (sequence 5′-CAGCUUUGGCUGAGCGUAU-3′) were obtained from Ambion (Austin, Texas, USA).

Statistical analysis

All values were reported as means ± standard error of the mean unless otherwise specified. Comparisons between experimental and control groups were performed by two-tailed unpaired Student’s t test. The value of p < 0.05 was considered to be statistically significant. Half-maximal inhibitory concentration (IC₅₀) values were calculated by concentration–response (Sigmoidal fitting) with Origin-Pro software Version 7.5.

Results

Effects of BDE-47 on proliferation and motility of SH-SY5Y cells

The effects of BDE-47 on the viability of SH-SY5Y cells were assessed using the CCK-8 kit. As shown in Figure 1(a), BDE-47 less than 10⁻³ M did not significantly (p > 0.05) alter the proliferation of SH-SY5Y cells for either 48 h or 72 h. However, treatment with 10⁻⁷ M and 10⁻⁵ M bisphenol A (BPA) obviously increased wound closure as compared to the control group (Figure 1(b)). The quantitative data in Figure 1(c) revealed that BDE-47 can promote the migration of SH-SY5Y cells in a dose-dependent manner. Our results suggested that BDE-47 may have limited cytotoxicity effects to human neuroblastoma SH-SY5Y cells, while it can increase their motility.

Figure 1.

Effect of BDE-47 on proliferation and motility of SH-SY5Y cells. (a) Cells were treated with various concentrations (10⁻¹⁰ to 10⁻³ M) of BDE-47 for 48 and 72 h and then cell viability was assessed using CCK-8 kit assay. (b) Representative images of wounds at 0 and 72 h in the presence or absence of BDE-47 (10⁻⁹ M, 10⁻⁷ M, and 10⁻⁵ M). Confluent monolayers of SH-SY5Y cells were scraped using a pipette tip to generate wounds and then were cultured in the presence or absence of BDE-47. (c) Quantitative analysis of wound-healing assay for SH-SY5Y cells treated with BDE-47. Data are presented as means ± SD of three independent experiments. *p < 0.05: compared with control; **p < 0.01: compared with control. BDE-47: 2,2′,4,4′-tetrabromodiphenyl ether; CCK-8: cell counting kit-8.

BDE-47 triggers the in vitro migration and invasion of SH-SY5Y cells

Boyden chambers were used to confirm the roles of BDE-47 on the in vitro migration and invasion of SH-SY5Y cells. As shown in Figure 2(a), the number of migrated cells was approximately 1.3- and 1.6-fold greater in cells treated with 10⁻⁷ M and 10⁻⁵ M BDE-47 for 72 h, respectively, than that of the control cells. The number of invaded cells was about 1.5- and 1.9-fold greater in cells treated with 10⁻⁷ M and 10⁻⁵ M BDE-47 for 72 h, respectively, than that of the control cells. The results further supported that BDE-47 can significantly promote the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells.

Figure 2.

BDE-47 triggers the in vitro migration and invasion of SH-SY5Y cells. Cells were allowed to migrate trans well chambers or spread through the matrix gel and into the underside of the filter for 48 h and 72 h in the presence or absence of BDE-47 (10⁻⁷ or 10⁻⁵ M). The migrated or invasive cells were fixed, stained, and photographed. The number of migrated (a) or invaded (b) cells were compared with the control. (c) Western blot analysis showed that the expression of cell motility-related proteins E-Cad, ZO-1, MMP-9, FN, N-Cad, and Vim in SH-SY5Y cells exposed to 10⁻⁷ or 10⁻⁵ M BDE-47 for 72 h. (d) Real-time PCR showed the mRNA expression of cell motility-related proteins E-Cad, ZO-1, MMP-9, FN, N-Cad, and Vim in SH-SY5Y cells exposed to 10⁻⁷ or 10⁻⁵ M BDE-47 for 48 h. Data represent the average of three independent experiments. *p < 0.05: compared with control; **p < 0.01: compared with the control. BDE-47: 2,2′,4,4′-tetrabromodiphenyl ether; E-Cad: E-cadherin; ZO-1: zona occludin-1; MMP-9: matrix metalloproteinase-9; FN: fibronectin; N-Cad: N-cadherin; Vim: vimentin; PCR: polymerase chain reaction; mRNA: messenger RNA.

Western blot analysis revealed that both 10⁻⁷ M and 10⁻⁵ M BDE-47 can downregulate the epithelial makers E-Cad and ZO-1, while upregulate the MMP-9 in SH-SY5Y cells treated for 72 h. BDE-47 had no obvious effect on the expression of FN, N-Cad, and Vim (Figure 2(c)). This was supported by the results of real-time PCR, which showed that BDE-47 treatment significantly downregulated the mRNA levels of E-Cad and ZO-1 while upregulated that of MMP-9 (p < 0.05, Figure 2(d)). Collectively, these results indicated that BDE-47 plays an important role in promoting in vitro migration and invasion of SH-SY5Y cells and modulates the expression levels of motility-related molecules.

MMP-9 is essential for BDE-47-induced migration of SH-SY5Y cells

In order to verify the roles of motility-related molecules in BDE-47-induced migration of cells, SH-SY5Y cells were transfected with nontargeting control si-RNA or MMP-9-specific si-RNA (si-MMP-9) for 24 h and then treated with BDE-47 for another 72 h. Both real-time PCR and Western blot analysis revealed that MMP-9 was successfully silenced by si-MMP-9 (Figure 3(a) and (b)). Further, BDE-47 significantly upregulated the mRNA and protein levels of MMP-9 in the presence of negative control si-RNA (si-NC) while did not attenuate si-MMP-9’s downregulation of MMP-9 in SH-SY5Y cells.

Figure 3.

MMP-9 is essential for BDE-47-induced migration of SH-SY5Y cells. SH-SY5Y cells pretreated with or without MMP-9-specific si-RNA or negative control si-RNA for 15 min were further treated with or without 10⁻⁵ M BDE-47 for 24 h, and then the mRNA (a) and protein (b) levels of MMP-9 were detected by real-time PCR and Western blot analysis, respectively. Further, the in vitro migration (c) and invasion (d) of SH-SY5Y cells were measured by trans well after treated with 10⁻⁵ M BDE-47 for 72 h. Data represent the average of three independent experiments. *p < 0.05: compared with control; **p < 0.01: compared with the control. MMP-9: matrix metalloproteinase-9; BDE-47: 2,2′,4,4′-tetrabromodiphenyl ether; si-MMP: matrix metalloproteinase-9-specific small interfering RNA; si-RNA: small interfering RNA; si-NC: negative control small interfering RNA; mRNA: messenger RNA; PCR: polymerase chain reaction.

Then, the in vitro migration and invasion of SH-SY5Y cells were assessed in the presence of si-MMP-9. The results revealed that silence of MMP-9 significantly suppressed the in vitro migration and invasion of SH-SY5Y cells. It also successfully abolished the 10⁻⁵ M BDE-47-induced migration and invasion of cells. While si-NC did not impair the promoting effects of 10⁻⁵ M BDE-47 on cell motility (Figure 3(c) and (d)). Generally, our results revealed that MMP-9 is essential for BDE-47-induced migration of SH-SY5Y cells.

GPER/PI3K/Akt mediated the BDE-47-induced cell migration

It was reported that the endocrine-disrupting effects of environmental estrogens were mediated by classic estrogen receptor α/β (ER α/β), GPER, and estrogen-related receptors (ERRs).²⁵ Therefore, the roles of GPER, ER α/β, and ERR α in BDE-47-induced MMP-9 were investigated by adding their specific antagonists. The results showed that G15, but either ICI 182,780 or XCT-790, significantly abolished the 10⁻⁵ M BDE-47-induced upregulation of MMP-9 (Figure 4(a)). It suggested that GPER, rather than ER α/β or ERR α, mediated the BDE-47-induced upregulation of MMP-9.

Figure 4.

GPER/PI3K/Akt mediated the BDE-47-induced migration of SH-SY5Y cells. (a) SH-SY5Y cells pretreated with G15 (a specific antagonist of GPR30, 1 μM) ICI 182,780 (ICI, a specific antagonist of ER α/β, 1 μM), or XCT-790 (XCT, a specific antagonist of ERR α, 1 μM), BDE-47 (10⁻⁵ M) alone or BDE-47 after a 90-min pretreatment with ICI 182,780 or G15 for 72 h. Then, the protein levels of MMP-9 were measured by Western blot analysis; SH-SY5Y cells pretreated with AG1478 (a potent antagonist of EGFR, 10 μM), LY294002 (inhibitor of PI3K/Akt, 10 μM), PD 98059 (an ERK1/2 antagonist, 10 μM), SB 203580 (inhibitor of p38-MAPK, 10 μM) for 30 min and then exposed to 10⁻⁵ M BDE-47 for another 72 h. The expression of MMP-9 was measured by Western blot analysis (b), and the in vitro migration of SH-SY5Y cells were measured by trans well (e). (c) SH-SY5Y cells were treated with 10⁻⁵ M BDE-47 for the indicated times, and then the p-Akt and Akt were measured by Western blot analysis; (d) SH-SY5Y cells were treated with various concentrations of BDE-47 for 30 min, and then the p-Akt and Akt were measured by Western blot analysis. Data represent the average of three independent experiments. **p < 0.01: compared with control. GPER: protein-coupled estrogen receptor 1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; Akt: protein kinase B; GPR30: G protein-coupled receptor 30; ERα/β: estrogen receptor α/β; ERRα: estrogen-related receptor; EGFR: epidermal growth factor receptor; p38-MAPK: p38-mitogen-activated protein kinase; p-Akt: phospho-protein kinase B.

To investigate the downstream signals of GPER responsible for the BDE-47-induced cell migration, inhibitors of epidermal growth factor receptor (EGFR; AG1478), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt (LY294002), mitogen-activated protein kinases (MAPK; PD98059), and p38 (SB-203580) were used since previous studies reported that GPER can activate these molecules and activation of these pathways can modulate the MMP-9.²⁶ We found that LY294002, rather than other inhibitors, obviously abolished the BDE-47-induced upregulation of MMP-9 in SH-SY5Y cells (Figure 4(b)). In addition, BDE-47 treatment increased the phosphorylation of Akt of SH-SY5Y cells in both time- (Figure 4(c)) and concentration- (Figure 4(d)) dependent manners. The phosphorylation of Akt was rapidly observed in SH-SY5Y cells after treated with 10⁻⁵ M BDE-47 for 5 min. Furthermore, G-15 and LY294002, rather than other inhibitors, significantly (p < 0.05) attenuated the BDE-47-induced in vitro migration of SH-SY5Y cells (Figure 4(c)). Taken together, our results suggested that GPER/PI3K/Akt is the signal pathway responsible for BDE-47-induced migration of human neuroblastoma SH-SY5Y cells and upregulation of MMP-9.

Discussion

There is increasing concern regarding PBDE global distribution and impact upon life. Laboratory and epidemiological studies showed that exposure to PBDEs may be associated with a wide range of biological deviations, including carcinogenesis, neurological changes, reproductive and behavioral abnormalities, and dysfunctions of the immune system.²⁷ BDE-47 is the most abundant PBDE congener in human tissues such as blood in children.²⁸ Although BDE-47 is suggested to be neurotoxic, investigation into the neurotoxicity and carcinogenicity of BDE-47 in humans remains surprisingly limited and uncertain. This study revealed for the first time that BDE-47 significantly promoted the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells via the upregulation of MMP-9 and GPER/PI3K/Akt signals mediated this process.

This study revealed that BDE-47 less than 10⁻³ M had limited effects on the proliferation of SH-SY5Y cells. Recent study also indicated that micromolar BDE-47 did significantly inhibit the proliferation of SH-SY5Y cells; however, the combination of BDE-47 and PCB-153 had significantly greater cytotoxicity on SH-SY5Y cells than BDE-47 alone.²⁹ Recent study also suggested that other PBDE congeners had limited effects on cell viability. For example, the study of Slotkin et al. also showed that growth of PC12 cells was unimpaired by BDE99 at 50 μM.³⁰ However, other studies also revealed that BDE-47 can increase the reactive oxygen species production, activate the IRE1 pathway,³¹ activate the p53-dependent mitochondrial apoptotic pathway,³² and then inhibit the proliferation of SH-SY5Y. Generally, the contradictory data suggested that further studies are needed to illustrate the effects of BDE-47 on the cell viability of human neuroblastoma SH-SY5Y cells.

Interestingly, our data suggested that BDE-47 can promote the motility of SH-SY5Y cells in time- and concentration-dependent manners. There are limited published data about the role of PBDE on the invasion of cancer cells. One previous study suggested that about one-third (32 of 111) of the identified proteins in the neonatal cortex after PBDE-99 exposure for 24 h are cytoskeleton related, including cofilin, β-actin, and microtubule-associated protein tau.³³ Another study indicated that 3 weeks of PBDE-47 exposure reduced the levels of the microtubule subunit β-tubulin in the mussel Mytilus edulis.³⁴ All these data supported that BDE-47 has the potential ability to trigger the metastasis of human neuroblastoma cells.

In this study, we found that BDE-47 treatment significantly downregulated the epithelial makers E-Cad and ZO-1, while upregulated the MMP-9 at both the mRNA and protein levels in SH-SY5Y cells. E-Cad and ZO-1 are the main components of extensive adheren junctions in epithelial cells and the disappearance of which is the hallmark for metastasis of cancer cells.³⁵ MMPs are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development and cell migration.³⁶ MMP-9, which is the key effector of ECM remodeling, is thought to be important in metastasis.³⁷ Generally, the modulation of metastasis-related molecules further supported the migration promotion effects of BDE-47 on SH-SY5Y cells.

Considering that MMP-9 can trigger the migration and invasion of various types of cancer cells,³⁸ the upregulation of MMP-9 is associated with poor prognosis of human neuroblastoma.³⁹ To verify the roles of MMP-9 in BDE-47-induced migration of SH-SY5Y cells, it was successfully silenced by specific si-RNA. The results revealed that si-MMP-9 significantly attenuated the BDE-47-induced upregulation of MMP-9 and in vitro migration and invasion, which suggested that MMP-9 is essential for BDE-47-induced metastasis of SH-SY5Y cells. To our knowledge, this is the first study that PBDE can upregulate the MMP-9 and then promote the migration of cancer cells. While for other kinds of environmental estrogens, previous studies indicated that BPA-induced migration of OVCAR-3 ovarian cancer cell was accompanied by upregulation of the migration-related factors MMP-2 and MMP-9.⁴⁰ BPA was also reported to stimulate the secretion of MMP-9 in human granulosa–lutein cells.⁴¹ Therefore, MMP-9 might be a target for the environmental estrogens triggering the metastasis of cancer cells.

Estrogen signaling is mediated through the soluble nuclear receptors ER α, ER β, ERR α, and membrane GPER, a seven-transmembrane-spanning G protein-coupled receptor.⁴² In this study, GPER, while either ER α/β or ERR α, mediated the biological effects of BDE-47 since antagonist of GPER (G15) obviously attenuated the BDE-47-induced upregulation of MMP-9, but ICI 182 780 or XCT-790, the specific inhibitor of ER α/β and ERR α, respectively, had no effects. This was similar with the recent studies that GPER mediates the BPA-induced rapid activation in breast cancer cells⁴³ and testicular seminoma cells.⁴⁴ GPER can also mediate rapid signaling events through activation of MAPK, PI3K/Akt, Src kinase, and other related pathways.⁴⁵ In this study, we found that LY294002, the inhibitor of PI3K/Akt, significantly abolished the BDE-47-induced MMP-9 upregulation and in vitro migration of SH-SY5Y cells. Furthermore, BDE-47 increased the phosphorylation of Akt in SH-SY5Y cells in both time- and concentration-dependent manners. PI3K/Akt has been reported to promote the metastasis of various cancer cells.^24,46 In the endometrial cells, PI3K/Akt signaling pathway is involved in 17β-estradiol-induced migration of endometrial cells.⁴⁷ The activation of PI3K/Akt can cause upregulation of MMP-9 in human tracheal smooth muscle cells⁴⁸ and hepatocellular carcinoma.⁴⁹ This might be mediated by the activation of mammalian target of rapamycin (mTOR)⁴⁹ or nuclear factor-κB^48,50
–52 by PI3K/Akt signals and needs further studies. Generally, our study revealed that GPER/PI3K/Akt signals mediated the upregulation of MMP-9 and promotion of cell migration of human neuroblastoma SH-SY5Y cells induced by BDE-47.

In conclusion, this study characterized that BDE-47 has limited effects on the cell viability but can significantly promote the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells via upregulation of MMP-9 in dose- and time-dependent manners. Further, GPER/PI3K/Akt signals mediated the BPA-induced cell migration and upregulation of MMP-9. This study provided new insight for the first time that the metastasis of human neuroblastoma cells is stimulated by BDE-47 and possibly by other PBDE congeners. The effects of BDE-47 on normal and other cancer cells remain to be determined.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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