Microcystin-LR Promotes Melanoma Cell Invasion and Enhances

Article pubs.acs.org/est

Microcystin-LR Promotes Melanoma Cell Invasion and Enhances Matrix Metalloproteinase-2/‑9 Expression Mediated by NF-κB Activation Xu-Xiang Zhang,†,§ Ziyi Fu,‡,§ Zongyao Zhang,† Chen Miao,‡ Pengfei Xu,‡ Ting Wang,‡,* Liuyan Yang,† and Shupei Cheng† †

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, China ‡ State Key Laboratory of Reproductive Medicine, Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China S Supporting Information *

ABSTRACT: This study aimed to explore the molecular mechanisms behind the stimulation effects of microcystin-LR (a well-known cyanobacterial toxin produced in eutrophic lakes or reservoirs) on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Boyden chamber assay showed that microcystin-LR exposure (>12.5 nM) evidently enhanced the invasion ability of the melanoma cells (MDA-MB-435). Tumor Metastasis PCR Array demonstrated that 24 h microcystin-LR treatment (25 nM) caused overexpression of eight genes involved in tumor metastasis, including MMP-2, MMP-9, and MMP-13. Quantitative real-time PCR, Western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after microcystin-LR exposure (P < 0.05 each). Immunofluorescence assay and electrophoretic mobility shift assay revealed that microcystin-LR could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus and enhancing NF-κB binding ability. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that microcystin-LR can act as a NF-κB activator to promote MMP-2/-9 expression and melanoma cell invasion, which deserves more environmental health concerns.

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INTRODUCTION

which might cause the majority of cancer treatment failure and patient death.14 Tumor metastasis occurs by a series of steps including vessel formation, cell attachment, invasion and cell proliferation.15 Tumor cells must move through and degrade surrounding tissue barriers to escape the primary site and colonize secondary organs, so the degradation of basement membranes and extracellular matrix (ECM) is a crucial step for tumor metastasis.16 This process requires different cellular proteolytic enzymes, among which matrix metalloproteinases (MMPs) are one of important families of proteinases responsible for the ECM destruction.17 Among over 20 identified MMPs, MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) can efficiently degrade native collagen types IV and V, fibronectin, entactin, and elastin.18 Therefore, the two proteases are considered crucial for cell invasion, and MMP-2/-9 overexpression is closely related to poor prognosis in patients.19

Cyanobacteria frequently form water blooms in eutrophic lakes or reservoirs and often produce various toxins. As a common group of cyanobacterial toxins,1 microcystins include approximately 80 known structural variants.2 Microcystin-LR is a cyclic heptapeptide,3 which can be accumulated in aquatic wildlife4 and transferred to higher trophic levels with the risk of livestock and human poisoning.5,6 It is well-known that microcystin-LR has tumor-promoting activity in humans.7 Epidemiologic studies have demonstrated that microcystins in contaminated water may contribute to the higher incidences of primary human liver8 and colon9 carcinomas. Recently, great efforts have been made to investigate possible mechanisms behind microcystins-induced carcinogenesis, focusing on the induction of reactive oxygen species involved in subsequent DNA damage,10 chromosomal aberrations,11 and alteration of proto-oncogenes12 or antioncogenes13 expression profiles, which are closely related to cell apoptosis and survival. However, information about the relationship between microcystin-LR exposure and tumor metastasis is limited, although invasion is an important event during carcinoma progression, © 2012 American Chemical Society

Received: Revised: Accepted: Published: 11319

December 24, 2011 September 13, 2012 September 14, 2012 September 19, 2012 dx.doi.org/10.1021/es3024989 | Environ. Sci. Technol. 2012, 46, 11319−11326

Environmental Science & Technology

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ECM gel before they were air-dried overnight at room temperature. Approximately 4 × 104 cells were added to the upper chamber and cultured in L15 without CBS, and L15 with 10% CBS was added to the lower chamber. For drug inhibition experiments, JSH-23 (10 μM) was added to the upper filters prior to invasion. During 72 h incubation at 5% CO2 and 37 °C, cells crossed the ECM and adhered to the opposite surface of the filter. The filters were then fixed with 4% paraformaldehyde and stained with crystal violet before the cells on the upper surface were removed completely with a cotton swab. The cell migration from the upper to the lower side of the filter was observed under light microscopy at a magnification of ×100. Cells were then lysed by extraction buffer (Millipore, ECM210) and collected for the detection of optical density (OD) at 570 nm. OD value of the treated cells was normalized to the control (no microcystin-LR treatment). Tumor cell invasiveness was defined as the average OD of the treated cells. PCR Array of Tumor Metastasis-Related Genes. In order to explore the molecular mechanisms of the cell invasion stimulated by microcystin-LR exposure, we used the Tumor Metastasis RT2 Profiler PCR Array (catalog no. PAHS-028A) (SABiosciences) to investigate the expression changes of 84 genes known to be involved in tumor metastasis. These genes selected for this array encode several classes of protein factors related to tumor metastasis (http://www.sabiosciences.com/ rt_pcr_product/HTML/PAHS-028A.html). Cells treated with 25 nM microcystin-LR for 24 h were chosen to investigate the expression of the metastasis-related genes, since the cells were most sensitive to microcystin-LR exposure in cell invasion assay. Five housekeeping genes, a genomic DNA control and three positive controls were included to ensure high quality data normalization across samples. Cycle threshold (CT) value obtained for the gene expression was normalized to the average CT of the different housekeeping genes. PCRs were conducted in duplicate for both the treated and the control cells, and the fold change of gene expression was determined following Livak and Schmittgen.31 To ensure statistical significance, the differentially expressed genes were identified as the ones with fold change beyond ±2.0.32 RNA Extraction and Quantitative Real-Time PCR. The mRNA expression of MMP-2/-9 genes (mmp-2/-9) were quantified using real-time reverse transcription PCR (qRTPCR) with β-actin as the housekeeping gene. After cells were treated with microcystin-LR, total RNA was isolated from melanoma cells with TRIZOL reagent according to the manufacturer’s protocol (TaKaRa, Japan). The purified total RNA (1 μg) was then reverse-transcribed using the First Strand cDNA Synthesis Kit (TaKaRa, Japan). Total RNA and cDNA concentrations were determined by a biophotometer (Eppendorf, Germany) and the obtained cDNA was kept at −20 °C until further analyses. Each reaction was conducted in 96-well plates with a final volume of 20 μL including 10 μL of SYBR Green PCR Master Mix (TaKaRa, Japan), plus 1 μL of each primer (2 μM) (Supporting Information (SI) Table S1) and 8 μL of template DNA. Thermal cycling and fluorescence detection were conducted on a Corbett Rotor-GeneTM RG65H0 (Corbett, Australia) using the following protocol: 94 °C for 2 min, followed by 40 cycles of 94 °C for 20 s, 55 °C for 20 s and 72 °C for 40 s. Each reaction was run in triplicate. The expression levels of mmp-2/-9 genes were normalized to βactin levels according to Livak and Schmittgen.31 After electrophoresis of PCR products, no primer-dimer was

MMPs expression is often mediated by activating nuclear factor kappaB (NF-κB).20 NF-κB is an important transcription factor that controls several cellular responses including inflammation,21 apoptosis,22 survival23 and invasion.24 Activation of NF-κB appears to occur via phosphorylation of endogenous inhibitors (IκB) of NF-κB.23 Once the IκB proteins are phosphorylated and subsequently degraded, the NF-κB complex may thereby be released for nuclear translocation and bind to DNA element, resulting in expression of target genes.25 Although previous studies have indicated that NF-κB activation contributes to the enhanced invasiveness of carcinoma cells26 by regulating MMP-927 and MMP-228 expression, the effects of environmental pollutants on cancer cell invasion via this pathway are still unknown. Microcystin-LR can inhibit PP1/PP2A (Ser/Thr phosphatases) activity10,11 and suppress the dephosphorylation of downstream proteins, such as protein kinase Akt and mitogenactivated protein kinase p38, which are involved in cell invasion.15,16 Moreover, our previous study showed that hepatic MMP-2/-9 expression was evidently enhanced in the mice after oral administration of microcystin-LR for 90 days.29 In this study, different types of cell lines (including hepatocarcinoma cells, gastric cancer cells, and melanoma cells) were first used to determine whether microcystine-LR can enhance carcinoma cells invasion. Among the cells lines, MDA-MB-435 melanoma cells showed the highest invasiveness after microcystin-LR treatment, so we further explored the role of NF-κB in the underlying molecular mechanism mediating the overexpression of MMP-2/-9 and enhancement of invasiveness of the melanoma cells exposed to microcystin-LR.

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EXPERIMENTAL SECTION Reagents and Antibodies. Microcystin-LR (purity ≥95%, by HPLC) was purchased from Alexis Biochemicals (Switzerland). Human melanoma cell MDA-MB-435, hepatocarcinoma cell HepG2 and gastric cancer cell SGC7901 were obtained from the American Type Culture Collection. Cell culture medium L15 was purchased from Gibco (Pufei Biotechnology Co., China). All antibodies used in this study were purchased from Cell Signaling Technology. Other reagents were purchased from Sigma−Aldrich Inc. unless otherwise mentioned. Cell Culture and Exposure. MDA-MB-435 cells were maintained in L15 medium (Gibco), and HepG2 and SGC7901 cells were cultured in DMEM (Hyclone) medium containing 100 U/mL streptomycin and 100 U/mL penicillin supplemented with 10% calf bovine serum (CBS). Cells were cultured in a humidified atmosphere of 5% carbon dioxide (CO2) at 37 °C. Microcystin-LR was dissolved in sterile water to make a 0.25 mM stock solution and then added in the medium to reach different doses (0, 1, 5, 12.5, 25, 50, 100, 200, 500, and 1000 nM). In order to explore the role of NF-κB in the molecular pathway mediating the promoted cell invasion and MMP-2/-9 expression, NF-κB inhibitor 4-methyl-N1-(3-phenyl-propyl)benzene-1,2-diamine (JSH-23) was dissolved in dimethyl sulfoxide (DMSO) and added to the medium at a final concentration of 10 μM.30 Cell Invasion Assay. After microcystin-LR exposure, Matrigel chamber assays were conducted to investigate the invasiveness of hepatocarcinoma cell line HepG2, gastric cancer cell line SGC7901 and melanoma cell line MDA-MB-435 using 24-well ECM-coated transwell plates. Briefly, the upper surface of the filter (8.0 μm in pore size) was coated with 40 μg of 11320

dx.doi.org/10.1021/es3024989 | Environ. Sci. Technol. 2012, 46, 11319−11326


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observed for both mmp-2/-9 and β-actin, and the specificity of the products was confirmed by melt curve analysis. Western Blotting. After the cells were lysed, a standard Western blotting analysis was conducted as previously described33 to investigate MMP-2/-9 and NF-κB activity. Briefly, nuclear and cellular protein extracts were prepared at the indicated time point using KEYGEN Protein Extraction Kit (KEYGEN, China). Protein lysates were then boiled in sodium dodecyl sulfate (SDS)-sample buffer for 5 min before SDSpolyacrylamide gel electrophoresis (8%) and transfer to polyvinylidene fluoride membranes (Bio-Rad). Membranes were blocked for 2 h in 5% milk-Tris-Buffered Saline Tween20 (TBST) at room temperature and incubated with the monoclonal antibodies (Cell Signaling Technology) of anti-βactin, anti-MMP-2, anti-MMP-9, or anti-NF-κB. Membranes were washed four times in TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Blots were visualized by enhanced chemiluminescence (Thermo) and analyzed using a scanning densitometer of BioSens Gel Imaging System (BIOTOP, China). Gelatin Zymography. MMP-2/-9 activity was analyzed by gelatin zymography. Briefly, cells (4 × 104) were seeded into a 24-well plate to adhere for 6−8 h in the presence of serum. Subsequently, the culture medium was replaced by L15 medium (400 μL per well) containing 2.5% CBS and the different concentrations of microcystin-LR. After incubation, the medium in each well was gathered and centrifuged for 10 min at 2000 rpm under 4 °C to remove cell debris. The supernatant was collected and mixed with loading buffer containing no dithiothreitol (DTT) before they were added in a nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis column containing 1 mg/mL gelatin. After electrophoresis, the gels were soaked twice in 2.5% Triton-X100 for 30 min to remove SDS, and then transferred to bath buffer containing 50 mM Tris (pH 8.0), 5 mM CaCl2 and 2 mM ZnCl2. After bath at 37 °C for 20 h, the gel was then stained with 0.1% Coomassie blue in 45% methanol and 10% acetic acid. The visualized bands indicating the presence of a protein with gelatinolytic activity were analyzed by BioSens Gel Imaging System (BIOTOP, China). Indirect Immunofluorescence and Confocal Laser Scanning Microscopy. A standard immunostaining procedure was employed to analyze NF-κB nuclear translocation activity. After 24 h treatment with microcystin-LR of 0, 5, 12.5, 25, and 50 nM, cells grown on thick slides were washed with phosphate buffered saline (PBS), fixed by immersion at room temperature with 4% polyformaldehyde for 20 min, and permeabilized with 0.1% Triton-X-100 in PBS at 4 °C for 10 min. Slides were then washed with PBS and blocked with blocking buffer consisting of 4% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cells were then incubated with primary anti-NF-κB monoclonal antibody (Cell Signaling Technology) (diluted 1:25) in blocking buffer overnight at 4 °C, followed by incubation with the secondary antirabbit fluorescein isothiocyanate (FITC) labeled antibody (Cell Signaling Technology) (diluted 1:100) in blocking buffer at room temperature for 1 h. Subsequently, cells were stained with 5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 2 min and washed with PBS. Coverslips and stained cells were captured and analyzed by a confocal laser scanning microscopy system (×400) (LSM710, Carl Zeiss, Germany). Control samples with horseradish peroxidase-conjugated secondary

antibody containing no DAPI showed a faint background staining (data not shown). EMSA Analysis. The cells treated with different doses of microcystin-LR were separately collected by centrifugation, and nuclear proteins were extracted using Cellular and Nuclear Protein Extraction Kit (PIERCE, Italy). Cells treated with 10 ng/mL TNFα for 60 min were prepared as positive control.34 Nuclear protein (10 μg) was used for electrophoretic mobility shift assay (EMSA) to detect the κB binding activity of NF-κB/ Rel using digoxigenin (DIG)-ddUTP-labeled double stranded oligonucleotide κB probe with sequence of 5′- AGT TGA GGG GAC TTT CCC AGG C -3′. The κB binding activity of NFκB/Rel was detected using EMSA Kit (Roche, Switzerland) according to the manufacturer’s protocol. For supershift assay, anti-NF-κB antibody was added during preincubation. The visualized bands were analyzed by BioSens Gel Imaging System (BIOTOP, China). For competition experiment, 100-fold specific oligonucleotide competitor (unlabeled probe) was added to the binding mixture and the reaction continued 10 min before addition of the labeled probe. Statistical Analysis. The differences in the invasion rate and protein expression between the treated cells and the control were determined using the ANOVA test. A p-value of less than 0.05 was considered statistically significant. Computerbased calculations were conducted using SPSS version 11.5 (SPSS Inc., Chicago, IL).

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RESULTS Effects of Microcystin-LR Treatment on Cell Invasion. Matrigel invasion assays were carried out to investigate the invasiveness of the melanoma cells (MDA-MB-435), hepatocarcinoma cells (HepG2) and gastric cancer cells (SGC7901) after microcystin-LR treatment (25 nM). As shown in SI Figure S1, microcystin-LR could promote invasion of MDA-MB-435 cells and SGC7901 cells, and the melanoma cells were more sensitive to microcystin-LR exposure. Furthermore, we evaluate the survival and invasiveness of MDA-MB-435 cells treated with different concentrations of microcystin-LR. High-dose microcystin treatments (>200 nM) can induce apoptosis of the melanoma cells within short exposure time (3−12 h) (P < 0.05 each), but no cell growth inhibition was observed after treatment at low concentrations ( 0.05 each) (SI Figure S2). However, the low-dose exposure stimulated the invasion of the cancer cells stained with crystal violet (Figure 1A), which was further confirmed by determining the OD value after the translocated cells were lysed and gathered (Figure 1B). Microcystin-LR treatments at 12.5, 25, or 50 nM significantly increased the number of the translocated cells (p < 0.05 each), but treatment at 1 or 5 nM caused no change in cell invasion (p > 0.05 each). Compared with the control, cell invasiveness was increased by 1.4 and 1.8 fold after exposure at 25 and 50 nM, respectively. Microcystin-LR stimulated the cancer cell invasion in a dose-dependent manner (R2 = 0.87) (Figure 1B). mRNA Expression of Tumor Metastasis Related Genes. In order to investigate the potential mechanism of the invasiveness enhancement, the Tumor Metastasis PCR Array was used to evaluate mRNA expression changes of the 84 genes responsible for tumor metastasis. Most of the genes showed no differential expression after the melanoma cells were treated with 25 nM microcystin-LR (SI Figures S3 and S4). However, the exposure still caused significant increase in the expression of eight genes involved in extracellular matrix degradation (mmp-2, mmp-9, and mmp-13), cell adhesion 11321



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MMP-2/-9 Over-Expression in Microcystin-Treated Cells. To confirm the effect of the microcystin-LR treatment on MMP-2/-9 expression, we investigated the mRNA and protein levels of MMP-2/-9 in MDA-MB-435 cancer cells after 24 h treatment with different concentrations of microcystin-LR. qRT-PCRs demonstrated that microcystin-LR treatment upregulated mmp-2/-9 expression (Figure 2A), which agrees with

Figure 1. Effects of microcystin-LR exposure on invasiveness of melanoma cells (MDA-MB-435) examined by transwell chamber assay (A) and statistical analysis (B). (A) The cells were seeded in a 24-well ECM-coated transwell chamber and incubated at 37 °C for 72 h with different concentrations of microcystin-LR (0, 1, 5, 12.5, 25, and 50 nM). The invaded cells were stained with crystal violet and three randomly selected regions were captured under light microscopy (×100). One representative of the three independent experiments is shown. (B) Cells were lysed by extraction buffer before collection and the optical density (OD) were detected at 570 nm for each sample. The value represents the mean ± SD from three different experiments. *P < 0.05 compared with the control cells without microcystin-LR treatment.

(cadherin-11 and pinin), cell growth and proliferation (insulinlike growth factor 1 and interleukin 8 receptor β), and cell cycle regulation (retinoblastoma 1) (Table 1). No significant downregulation was observed for the mRNA expression of each gene in the treated cells.

Figure 2. Expression of MMP-2/-9 in MDA-MB-435 cells detected by qRT-PCR (A), Western blotting (B) and gelatin zymography (C). (A) The cells cultured in monolayer were treated with different concentrations of microcystin-LR for 24 h. mRNA levels of mmp-2/9 were quantified by qRT-PCR. The mRNA levels were normalized to the expression of β-actin. The value represents the mean ± SD from three independent experiments. (B) After 24 h culture with microcystin-LR, the cells were treated with SDS lysis buffer and the total cellular protein (40 μg/lane) was used for Western blotting with a specific anti-MMP-2 or anti-MMP-9 antibody. The membrane was probed with an anti-ß-actin antibody to confirm equal loading. The blots are representative of three independent experiments. (C) After 72 h microcystin-LR treatment, MDA-MB-435 cells were removed and the supernatants were used for SDS-PAGE gelatin zymography analysis of MMP-2/-9. The experiments were conducted in triplicate. *P < 0.05 compared with the control cells without microcystin-LR treatment.

Table 1. Differentially Expressed Genes in Melanoma Cell MDA-MB-435 after 24 h Exposure to Microcystin-LR (25 nM) GenBank ID

gene symbol

NM_001797

Cdh11

NM_000618

Igf1

NM_001557 NM_002427

Il8rb mmp-13

NM_004530

mmp-2

NM_004994

mmp-9

NM_002687

Pnn

NM_000321

Rb1

function cadherin 11, type 2, OBcadherin (osteoblast) insulin-like growth factor 1 (somatomedin C) interleukin 8 receptor, beta matrix metallopeptidase 13 (type III collagenase) matrix metallopeptidase 2 (gelatinase A, type IV collagenase) matrix metallopeptidase 9 (gelatinase B, type IV collagenase) pinin, desmosome associated protein retinoblastoma 1

fold change of up-regulation 2.7 3.5 2.2 3.1 2.3

the results of cDNA microassay (Table 1). MMP-9 mRNA expression showed no increase at 5, 12.5, and 25 nM but an increase at 50 nM, whereas MMP-2 mRNA level was higher at 25 nM than at 50 nM (P < 0.05). Western blotting showed that the protein levels of MMP-2/-9 were also increased after microcystin-LR treatments at over 12.5 nM (Figure 2B), which

2.3 2.2 2.0 11322



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Figure 3. Activation of NF-κB (p65) in melanoma cell line MDA-MB-435 examined by immunostaining analysis (A), Western blotting (B) and electrophoretic mobility shift analysis (C). (A) Immunostaining analysis was performed to examine the effects of microcystin-LR on NF-κB nuclear translocation. After 24 h microcystin-LR treatment, MDA-MB-435 cells on the coverslips were blocked with PBS containing 4% BSA and immunostained with anti-p65 antibody, and then stained with FITC-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). The stained cells were captured by a confocal laser scanning microscopy system using a 400× magnification. The fluorescence intensity of p65 in nuclei of each sample was calculated and analyzed. *P < 0.05 compared with the control cells without microcystin-LR treatment. (B) Nuclear and cellular proteins were separately extracted before the protein levels of cytosolic NF-κB and nuclear NF-κB were detected by Western blotting. The nuclear translocation activity of NF-κB was determined by the ratio of p65 (nucleus)/p65 (cytosol). (C) NF-κB DNA-binding activity of MDA-MB435 cells was examined by electrophoretic mobility shift analysis. Nuclear protein extraction was used to detect the κB binding activity of p65. Line 1: 100-fold unlabeled probe for competition experiment; Line 2: cells pretreated with 10 ng/mL TNFα for 60 min (positive control); Line 3: the control cells without microcystin-LR treatment; Lines 4−7: cells treated with microcystin-LR at different concentrations (5, 12.5, 25, and 50 nM, respectively); Line 8: nuclear protein extraction of the microcystin-LR (25 nM) treated cells incubated with labeled probe and anti-p65 antibody. The black arrow indicates the complex of DIG-ddUTP-labeled double stranded oligonucleotide κB probe and NF-κB. The gray arrow indicates the oligonucleotide κB probe labeled protein-p65 antibody complex.

Mediation of NF-κB in MMP-2/-9 Expression and Cell Invasion. Drug inhibition experiment was conducted to confirm the role of NF-κB activation in the MMP-2/-9 overexpression and cell invasiveness enhancement. After treatment with NF-κB inhibitor JSH-23 (10 μM), the cells exposed to microcystin-LR (25 nM) had lower invasiveness (p < 0.05) (Figure 4A), revealing that NF-κB activation may contribute to the stimulation effect of microcystin-LR on the cellular invasion. Furthermore, gelatin zymography demonstrated that MMP-2/-9 activity was inhibited in the cells treated with both JSH-23 and microcystin-LR (Figure 4B and SI Figure S7), suggesting that NF-κB mediated MMP-2/-9 overexpression stimulated by microcystin-LR.

was further supported by gelatin zymography analysis (Figure 2C and SI Figure S5). Results of qRT-PCR, Western blotting and gelatin zymography revealed that microcystin-LR exposure could evidently up-regulate both mRNA and protein expression of MMP-2/-9 in MDA-MB-435 cells, which may be subsequently involved in the cell invasion promotion. NF-κB Activation in Microcystin-Treated Cells. We further examined the effect of microcystin-LR treatment on NF-κB activity to explore the potential molecular mechanisms of MMP-2/-9 overexpression. Immunofluorescence assays showed that microcystin-LR exposure accelerated nuclear translocation of NF-κB (p65), and the treatment at 25 nM induced the most notable change in fluorescence density (P < 0.05) (Figure 3A). Subsequently, the cytosol and nuclear proteins were isolated from the microcystin-treated cells for Western blotting of p65 (Figure 3B). As a result of microcystin treatment, ratio of the nuclear/cytosolic level of NF-κB was significantly elevated from 0.32 ± 0.04 (the control) to 0.89 ± 0.12 (25 nM microcystin) (P < 0.05) (SI Figure S6), which agrees with the result of immunofluorescence assay (Figure 3A). Electrophoretic mobility shift analysis showed that NF-κB binding ability was enhanced after microcystin-LR treatment (P < 0.05) (Figure 3C). Similar to the results of Western blotting and immunofluorescence assays, the highest NF-κB binding ability was observed in the cells treated with 25 nM microcystin-LR (Figure 3C). Super shifted band detection showed that the oligonucleotide κB probe/nuclear protein/ anti-p65 antibody complex was produced as a shifted band (Figure 3C), suggesting the specific binding of the p65 protein in the treated cells with the κB probe.

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DISCUSSION This is the first study revealing that the environmental pollutant microcystin-LR can increase invasiveness of melanoma cells. Accumulating evidence has shown that microcystin-LR can act as a tumor initiator to induce DNA damages.35 However, little information is available about the effect of microcystin-LR on cancer cell invasion, although clinical concerns have focused on screening and developing drugs capable of cancer metastasis inhibition.36,37 Recent studies have indicated that cancer cell invasion can be promoted by some physicochemical factors including extracellular calcium,38 progesterone receptor,39 glutamate,40 and bacterial peptidoglycan,41 but the relationship between environmental pollution and tumor metastasis remains unknown. In the present study, the Tumor Metastasis PCR Array showed that microcystin-LR exposure caused significant 11323



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cell invasion ability. In this study, invasiveness of hepatocarcinoma cells (MDA-MB-435) and gastric cancer cells (SGC7901) was significantly increased after microcystin exposure. Considering that MDA-MB-435 showed more evidently increased invasiveness, we chose the melanoma cells to elucidate the molecular mechanism behind the MMPs overexpression. First, we examined the effect of microcystinLR on NF-κB (p65) activity since previous studies have demonstrated that microcystin-LR can activate NF-κB in cancer cells22 and MMPs can be regulated by PI3-K/Akt signaling pathway through NF-κB activation.47 NF-κB/IκB complex located in the cytoplasm in a latent form often transfers to the nuclear when IκB was phosphorylated by Akt,24 so we detected the changes of NF-κB activity by observing NF-κB nuclear translocation. Immunofluorescence assays showed that microcystin-LR exposure induced nuclear translocation of NF-κB, which is further confirmed by Western blotting. As a nuclear transcriptional factor, p65 needs to bind to DNA to activate the target gene expression.23 EMSA suggested that microcystin-LR treatment enhanced DNA binding ability of NF-κB, revealing that NF-κB activation might mediate MMP-2/-9 overexpression induced by microcystin-LR. This hypothesis is supported by Lankoff et al.11 indicating that tumor-promoting activity of microcystin-LR arises from its ability to affect cell viability mainly through inhibition of protein phosphatases PP2A and PP1 (PKB/Akt regulators). Additionally, NF-κB in myeloma cells can be activated through phosphorylation of IκB proteins by Akt, allowing for translocation of the released NF-κB to the nucleus.48 Therefore, microcystin-LR might activate PKB/Akt NF-κB pathway through inhibition of PP2A and PP1 and phosphorylation of IκB proteins. The proposed molecular mechanism was further confirmed by the JSH-23 suppression assay, a well-established NF-κB inhibition method for MDA-MB-435 cells.30 The increased MMP-2/-9 activity and invasiveness were inhibited by JSH-23 in the melanoma cells cultured with microcystin-LR, suggesting that NF-κB plays a crucial role in the MMP-2/-9 overexpression and invasiveness enhancement. Although NF-κB is an important transcription factor controlling cell apoptosis,22 this study showed that appropriate increase of NF-κB activity could promote MMPs expression and cancer cell invasion, but did not affect cell viability, which is supported by Huang et al.24 and Cheng et al.47 A NF-κB response element exits in the promoter region of the human mmp-9 gene,47 and inhibition of carcinoma migration and invasion occurs if MMP-9 expression is suppressed, which was mediated by inhibiting NF-κB-binding activity.49 It has been indicated that thioredoxin can augment mmp-9 transcription through NF-κB in human MDA-MB-231 cells.27 NF-κB can also mediate MMP-2 expression28 by stimulating MT1-MMP expression and activating pro-MMP2.50 Taken together, microcystin-LR can inhibit protein phosphatases and activate NF-kB to induce MMP-2/-9 overexpression, resulting in the melanoma cell invasion. Additionally, previous studies have shown that some extracellular compounds can interact with cell membrane receptor protein to disturb intracellular NF-κB activation and MMP-2/-9 expression, subsequently affecting cancer cell invasion and migration.28,38,51 Since OATPs were not detected in MDA-MB435 cells, extracellular microcystin-LR might interact with specific receptor proteins on cell membrane to affect NF-κB activity and MMPs expression in MDA-MB-435 cells. However,

Figure 4. Role of NF-κB in the microcystin-induced MMP-2/9 overexpression and cell invasiveness enhancement analyzed by transwell invasion assay (A) and gelatin zymography (B). MDA-MB435 cells were separately incubated with JSH-23 (10 μM), microcystinLR (25 nM) and JSH-23 (10 μM)/microcystin-LR (25 nM). (A) The invaded cells were stained with crystal violet and three randomly selected regions were captured under light microscopy (×100). One representative of three independent experiments is shown. The OD at 570 nm was calculated and analyzed for the stained cells of each sample. *P < 0.05 compared with the control. (B) The medium were centrifuged to remove the cells, and the supernatants were harvested for gelatin zymography of MMP-2/-9.

overexpression of eight genes which are involved in tumor metastasis. Among the differentially expressed genes, three were found to be responsible for extracellular matrix degradation (mmp-2, mmp-9, and mmp-13). It is well-known that MMPs overexpression is closely associated with tumor metastasis,16 so we assessed the effects of microcystin-LR treatment on the mRNA and protein expression by using qRT-PCR, gelatin zymography and Western blotting. Results consistently showed that microcystin-LR could enhance MMP-2/-9 expression in the melanoma cells, which is supported by our previous studies demonstrating that hepatic MMP-2/-9 expression were stimulated in mice after chronic exposure to microcystin-LR29 or cyanobacterial blooming lake water.42 Effects of microsystins on cancer cell survival and invasion are cell-, time-, and concentration-dependent, since previous studies have indicated the contrasting cellular responses to microsystin-LR exposure.43,44 Monk et al.45 transiently transfected HeLa cervical adenocarcinoma cells with plasmids carrying organic anion transporting polypeptides (OATPs) to facilitate intracellular access of microcystin-LR, revealing that microcystin could kill HeLa cells if the compound gained intracellular access. Unfortunately, this study showed that OATP1B1 and OATP1B3 were absent in MDA-MB-435 cells (SI Figure S8). Gan et al.46 indicated that long-term exposure (2−4 days) to low-concentration microsystin-LR could stimulate liver cancer cells proliferation. Our results suggested that low-dose treatments (200 nM) could suppress cell viability within short exposure time (3−12 h). In order to exclude the influence on cell survival, we chose the low doses (0−50 nM) to explore the effect of microcystin-LR on cancer 11324



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(5) Jasionek, G.; Zhdanov, A.; Davenport, J.; Blaha, L.; Papkovsky, D. B. Mitochondrial toxicity of microcystin-LR on cultured cells: Application to the analysis of contaminated water samples. Environ. Sci. Technol. 2010, 45, 2535−2541. (6) Rogers, E. D.; Henry, T. B.; Twiner, M. J.; Gouffon, J. S.; McPherson, J. T.; Boyer, G. L.; Sayler, G. S.; Wilhelm, S. W. Global gene expression profiling in larval zebrafish exposed to microcystin-LR and Microcystis reveals endocrine disrupting effects of cyanobacteria. Environ. Sci. Technol. 2011, 45, 1962−1969. (7) Hernandez, J. M.; Lopez-Rodas, V.; Costas, E. Microcystins from tap water could be a risk factor for liver and colorectal cancer: A risk intensified by global change. Med. Hypotheses 2009, 72, 539−540. (8) Ueno, Y.; Nagata, S.; Tsutsumi, T.; Hasegawa, A.; Watanabe, M. F.; Park, H. D.; Chen, G. C.; Chen, G.; Yu, S. Z. Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis 1996, 17, 1317−1321. (9) Zhou, L.; Yu, H.; Chen, K. Relationship between microcystin in drinking water and colorectal cancer. Biomed. Environ. Sci. 2002, 15, 166−171. (10) Ž egura, B.; Zajc, I.; Lah, T. T.; Filipič, M. Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis. Toxicon 2008, 51, 615− 623. (11) Lankoff, A.; Bialczyk, J.; Dziga, D.; Carmichael, W. W.; Gradzka, I.; Lisowska, H.; Kuszewski, T.; Gozdz, S.; Piorun, I.; Wojcik, A. The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor. Mutagenesis 2006, 21, 83−90. (12) Li, H.; Xie, P.; Li, G.; Hao, L.; Xiong, Q. In vivo study on the effects of microcystin extracts on the expression profiles of protooncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins. Toxicon 2009, 53, 169−175. (13) Takumi, S.; Komatsu, M.; Furukawa, T.; Ikeda, R.; Sumizawa, T.; Akenaga, H.; Maeda, Y.; Aoyama, K.; Arizono, K.; Ando, S.; Takeuchi, T. p53 Plays an important role in cell fate determination after exposure to microcystin-LR. Environ. Health Perspect. 2010, 118, 1292−1298. (14) Bartels, R. H.; van der Linden, Y. M.; van der Graaf, W. T. Spinal extradural metastasis: Review of current treatment options. CACancer J. Clin. 2008, 58, 245−259. (15) Chaffer, C. L.; Weinberg, R. A. A perspective on cancer cell metastasis. Science 2011, 331, 1559−1564. (16) Chiang, A. C.; Massagué, J. Molecular basis of metastasis. N. Engl. J. Med. 2008, 359, 2814−2823. (17) Birkedal-Hansen, H.; Moore, W. G.; Bodden, M. K.; Windsor, L. J.; Birkedal-Hansen, B.; DeCarlo, A.; Engler, J. A. Matrix metalloproteinases: A review. Crit. Rev. Oral Biol. Med. 1993, 4, 197−250. (18) McCawley, L. J.; Matrisian, L. M. Matrix metalloproteinases: Multifunctional contributors to tumor progression. Mol. Med. Today 2000, 6, 149−156. (19) Daniele, A.; Zito, A. F.; Giannelli, G.; Divella, R.; Asselti, M.; Mazzocca, A.; Paradiso, A.; Quaranta, M. Expression of metalloproteinases MMP-2 and MMP-9 in sentinel lymph node and serum of patients with metastatic and non-metastatic breast cancer. Anticancer Res. 2010, 30, 3521−3527. (20) Chandrasekar, B.; Mummidi, S.; Mahimainathan, L.; Patel, D. N.; Bailey, S. R.; Imam, S. Z.; Greene, W. C.; Valente, A. J. Interleukin18-induced human coronary artery smooth muscle cell migration is dependent on NF-kappaB- and AP-1-mediated matrix metalloproteinase-9 expression and is inhibited by atorvastatin. J. Biol. Chem. 2006, 281, 15099−15109. (21) Vermeulen, L.; De Wilde, G.; Notebaert, S.; Vanden Berghe, W.; Haegeman, G. Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit. Biochem. Pharmacol. 2002, 64, 963−970. (22) Feng, G.; Abdalla, M.; Li, Y.; Bai, Y. NF-κB mediates the induction of Fas receptor and Fas ligand by microcystin-LR in HepG2 cells. Mol. Cell. Biochem. 2011, 352, 209−219.

this hypothesis needs further experimental identification. Microcystin-LR is widely distributed in the cyanobacterial blooming lakes or reservoirs serving as source of drinking water for local residents,1 so our findings pointed the importance of assessing the induced public health risks in drinking water. In conclusion, this study indicates that microcystin-LR exposure can promote melanoma cell invasion by stimulating MMP-2/-9 mRNA and protein expression. Microcystin-LR is able to activate NF-κB by accelerating translocation into the nucleus and enhancing binding ability. NF-κB activation may be responsible for the MMP-2/-9 overexpression and cancer cell invasiveness enhancement. This is the first study revealing that NF-κB mediates the stimulation effects of microcystin-LR on MMP-2/-9 expression and cancer cell invasion. Future work may include confirmation of the stimulation effects through in vivo exposure and epidemiological investigation.

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ASSOCIATED CONTENT

* Supporting Information S

Real-time reverse transcription PCR primers (Table S1), effects of microcystin-LR on invasiveness of different cancer cells (Figure S1), effects of microcystin-LR on MDA-MB-435 cell viability (Figure S2), scatter plot of metastasis-related genes (Figure S3), fold change of metastasis-related genes (Figure S4), differential expression of MMP-2/-9 (Figure S5), translocation of NF-κB (p65) in cancer cells (Figure S6), role of NFκB in the MMP-2/-9 overexpression (Figure S7), OATP1B1 and OATP1B3 genes in MDA-MB-435 cells (Figure S8). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +86-25-86863153; e-mail: wtbiocell@hotmail. com. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Basic Research Program of China (2008CB418102), National Natural Science Foundation of China (21177062), the Key Laboratory Medicine of Jiangsu Province (XK201114), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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REFERENCES

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Microcystin-LR Promotes Melanoma Cell Invasion and Enhances

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