Promotion of Melanoma Cell Invasion and Tumor Metastasis by

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Promotion of Melanoma Cell Invasion and Tumor Metastasis by Microcystin-LR via Phosphatidylinositol 3‑Kinase/AKT Pathway Pengfei Xu,†,⊥ Xu-Xiang Zhang,‡,⊥ Chen Miao,† Ziyi Fu,† Zhengrong Li,† Gen Zhang,† Maqing Zheng,§ Yuefang Liu,∥ Liuyan Yang,‡ and Ting Wang†,* †

Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China § College of Pharmacy, Nanjing University of Technology, Nanjing 211816, China ∥ Department of Pathology, Nanjing Medical University, Nanjing 210029, China ‡

S Supporting Information *

ABSTRACT: Recently, we have indicated that microcystinLR, a cyanobacterial toxin produced in eutrophic lakes or reservoirs, can increase invasive ability of melanoma MDA-MB435 cells; however, the stimulatory effect needs identification by in vivo experiment and the related molecular mechanism is poorly understood. In this study, in vitro and in vivo experiments were conducted to investigate the effect of microcystinLR on invasion and metastasis of human melanoma cells, and the underlying molecular mechanism was also explored. MDAMB-435 xenograft model assay showed that oral administration of nude mice with microcystin-LR at 0.001−0.1 mg/kg/d posed no significant effect on tumor weight. Histological examination demonstrated that microcystin-LR could promote lung metastasis, which is confirmed by Matrigel chamber assay suggesting that microcystin-LR treatment at 25 nM can increase the invasiveness of MDA-MB-435 cells. In vitro and in vivo experiments consistently showed that microcystin-LR exposure increased mRNA and protein levels of matrix metalloproteinases (MMP-2/-9) by activating phosphatidylinositol 3-kinase (PI3-K)/AKT. Additionally, microcystin-LR treatment at low doses (≤25 nM) decreased lipid phosphatase PTEN expression, and the microcystin-induced invasiveness enhancement and MMP-2/-9 overexpression were reversed by the PI3-K/AKT chemical inhibitor LY294002 and AKT siRNA, indicating that microcystin-LR promotes invasion and metastasis of MDA-MB-435 cells via the PI3-K/AKT pathway.

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INTRODUCTION Microcystin-LR is a cyclic heptapeptide,1 which has tumorpromoting activity in humans.2 Epidemiological studies have demonstrated that microcystins in eutrophic water produced by cyanobacteria may contribute to a higher incidence of primary human carcinomas,3 and accumulating evidence has shown that microcystin-LR acts as a tumor initiator.4 Current studies focus on the possible mechanisms of microcystin-induced carcinogenesis.5,6 The 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).7 In addition, microcystin-LR also blocks the dephosphorylation of downstream proteins, such as the protein mitogen-activated protein kinase (MAPK) p388 and AKT,9 which not only involve cell proliferation and survival, but also mediate tumor invasion and metastasis. However, it is unknown whether microcystinLR is involved in modulation of invasion and metastasis phenotypes and genotypes in tumor cells. Invasion is a crucial step for tumor metastasis. In this process, tumor cell degradation of basement membranes and extrac© 2013 American Chemical Society

ellular matrix (ECM) results in these tumor cells moving through and degrading surrounding tissue barriers to escape the primary site and colonize secondary organs. Matrix metalloproteinases (MMPs) are a family of 24 secreted membrane-type proteases that mediate cell invasion, among which MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) are important proteinases responsible for degradation of basement membranes and ECM.10 The expression of MMP-2/-9 are positively regulated by cell signaling pathways, such as the phosphatidylinositol 3-kinase (PI3-K)/AKT pathway.11,12 PI3-K/AKT is an important pathway that controls several cellular responses, including apoptosis,13 survival14,15 and invasion.16 PTEN (phosphatase and tensin homologue) acts as a lipid phosphatase to dephosphorylate phosphatidylinositol(3-5)-trisphosphate (PIP), causing PI3-K/ AKT inactivation. PI3-K/AKT activation is closely related to Received: Revised: Accepted: Published: 8801

February 15, 2013 June 7, 2013 June 26, 2013 June 26, 2013 dx.doi.org/10.1021/es4007228 | Environ. Sci. Technol. 2013, 47, 8801−8808

Environmental Science & Technology

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Madison, WI) following the manufacturer’s instructions after 24 h of exposure to microcystin-LR, and luciferase activity was measured with Luciferase Assay Reagent 10-Pack (Promega). β-gal activity of the cells was measured as follows: 1 μL of Mg solution (0.1 M MgCl2, 4.5 M beta-mercaptoethanol), 22 μL of 4 mg/mL O-nitrophenol-β-D-galactoside solution (O-nitrophenyl-beta-D-galactopyranoside) (in 0.1 M phosphate buffer [pH 7.5]), and 57 μL of 0.1 M phosphate buffer (pH 7.5), were added into the obtained cell extract solution (20 μL) to reach a total volume of 100 μL. As a control, the optical density (OD) volume of β-gal at 450 mm was measured in parallel. To identify the reliability of LY294002 suppression assay, small interfering RNA (siRNA) was also used to inhibit AKT expression in MDA-MB-435 cells. The AKT siRNA interference method was described in detail in the Supporting Information (SI). Cell Invasion Assay. Matrigel chamber assays were conducted to investigate melanoma cancer cell invasion using 24-well ECM-coated transwell inserts. Briefly, the upper surface of the filter (8.0 μm in pore size) was coated with 40 μg ECM gel before being air-dried overnight at room temperature. Approximately, 4 × 104 cells were added to the upper chamber and cultured in L15 without CBS. L15 with 10% CBS was added to the lower chamber. Microcystin-LR (25 nM) was added to the upper filters prior to invasion. After 72 h of incubation in an environment of 5% CO2 at 37 °C, cells crossed the ECM and adhered to the opposite surface of the filters. 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. 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 using RIPA cell lysis buffer and collected for the detection of OD at 570 nm. Tumor cell invasiveness was defined as the mean OD volume of cells. Animals. Twenty-eight female athymic BALB/c mice (4−6 weeks old) obtained from the Laboratory Animal Center, Academy of Military Medical Science, were randomly divided into four groups (seven mice in each group). The animals were housed under specific pathogen-free-conditions. Nude mice were exposed to microcystin-LR separately at three different doses: low (0.001 mg/kg/d), medium (0.01 mg/kg/d) and high (0.1 mg/kg/d) by oral gavage. After 1 week, the MDAMB-435 cells (1 × 107) mixed with 0.15 mL of PBS were subcutaneously injected into the left upper flank region of each mouse with a 100-gauge needle. Body weights and tumor sizes were recorded once a week (tumor volume =ab2/2 in mm, where a and b are the longest and the shortest perpendicular diameters of the tumor, respectively). After 6 weeks of oral gavage, all nude mice were killed under deep anesthesia. The tumors were removed and photographed, and then used for multiple assays, including quantitative real-time PCR (qPCR) and immunohistochemistry (IHC). Three lung tissues of every nude mouse were randomly chosen for hematoxylin−eosin (H&E) staining, and the total number of lung metastasis was counted under a microscope (×200). The metastases were classified into four grades based on the number of tumor cells present at the section for each metastatic lesion: grade I, ≤20 tumor cells; grade II, 21−50 tumor cells; grade III, 51−100 tumor cells; and grade IV, >100 tumor cells.21,22 qPCR. Total RNA of tumor tissue samples was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The purified total RNA (1 μg) was then reversely

tumor progression, and PTEN expression is down-regulated or lost among highly malignant or last-stage metastatic cancer. Our previous study has revealed the stimulatory effect of microcystin-LR on cancer cell invasion and migration through in vitro experiment.17 However, in vivo evidence is still unavailable regarding microcystin-LR effect on tumor metastasis, even though metastasis is the major cause of treatment failure in cancer patients. It is known that microcystin-LR can increase MMP-2/-9 expression in cancer cells18 and mice liver19 and MMP-2/-9 overexpression is mediated by nuclear factor kappa B (NF-κB).17 PI3-K/AKT can phosphorylate endogenous inhibitors (IκB) of NF-κB releasing NF-κB to cell nucleus.20 However, role of PI3-K/AKT pathway in microcystin-induced MMPs overexpression remains unknown. This study aimed to assess the effects of microcystin-LR exposure on the invasion and metastasis of melanoma cells MDA-MB-435 through in vitro and in vivo experiments, and also to investigate the role of the PI3-K/AKT pathway in the underlying molecular mechanism. The results of this study are expected to extend our knowledge about the environmental health risks induced by microcystin-LR.

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MATERIALS AND METHODS Cell Culture. The MDA-MB-435 human melanoma cancer cell line was obtained from the American Type Culture Collection and maintained in monolayer culture in L15 medium (Gibco, Grand Island, NY) supplemented with 10% calf bovine serum (CBS) (Thermo, Waltham, MA), 100 U/ml streptomycin (Gibco), and 100 U/mL penicillin (Gibco). Cells were cultured under the conditions of 5% CO2 and 95% humidity at 37 °C. Western Blotting Assay. Cells were lysed with RIPA cell lysis buffer (Beyotime, China) containing 1 mM phenylmethylsulfonyl fluoride. Protein lysates were then boiled for approximately 5 min in sodium dodecyl sulfate sample buffer (Beyotime) before being subjected to 8% sodium dodecyl sulfate-polyacrylamide gel (40 μg protein/lane) electrophoresis and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA). Membranes were then blocked for 2 h in 5% milk-phosphate buffered saline with 0.1% Tween-20 to block nonspecific binding and immunoblotted overnight at 4 °C with the following primary antibodies: rabbit anti-pAKT (Thr308) (1:500, Cell Signal Technology, Danvers, MA), antiPTEN (1:1000, Bioworld, China), anti-MMP-2 (1:500, Cell Signal Technology), anti-MMP-9 (1:500, Cell Signal Technology), and anti-GAPDH (1:8000, Bioworld). Membranes were washed four times in 0.1% Tween-20 and incubated with the appropriate peroxidase-conjugated secondary antibodies (1:1000, Cell Signal Technology) for 2 h. Blots were visualized by Enhanced Chemiluminescence (Thermo) and analyzed using a scanning densitometer with molecular analysis software (BioSens Gel Imaging System, Biotop, China). Luciferase Assay. MDA-MB-435 cells were grown on a 24-well plate for 24 h to reach approximately 80% confluence prior to transfection. The cells in each well were cotransfected with 0.4 μg of mmp-2/-9-binding site-derived luciferase reporter plasmid and 0.1 μg of β-galactosidase (β-gal) expression plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. After transfection for 6 h, cells were treated with the PI3-K/AKT chemical inhibitor LY294002 (20 μM), microcystin-LR (25 nM) (Alex, Switzerland), or both LY294002 (20 μM) and microcystin-LR (25 nM). Cell lysates were prepared with Reporter Lysis Buffer (Promega, 8802

dx.doi.org/10.1021/es4007228 | Environ. Sci. Technol. 2013, 47, 8801−8808

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Figure 1. PI3-K/AKT activation in MDA-MB-435 cells by microcystin-LR. (A) Activation of AKT in MDA-MB-435 cells. Cells were treated with microcystin-LR (0, 5, 12.5, 25, or 50 nM) for 1 h. A total of 40 μg of cellular lysate from control and microcystin-treated cells was analyzed for the expression of pAKT. (B) Inhibition of PTEN in MDA-MB-435 cells. Cellular lysate derived from cells was treated with microcystin-LR at the indicated concentrations for 24 h. Values represent the mean ± SD from three different experiments. *p < 0.05 compared with control cells without microcystin-LR treatment.

Figure 2. Role of PI3-K/AKT in microcystin-LR-induced MMP-2/-9 overexpression and cell invasion promotion. (A) A luciferase assay was carried out as described in the Materials and Methods. After 6 h of transfection with MMP-2/-9 and β-gal plasmid, cells were treated with LY294002 (20 μM), microcystin-LR (25 nM) or LY294002 (20 μM)/microcystin-LR (25 nM) for 24 h. Cells were then lysed by reporter lysis buffer and luciferase activity was measured. The OD volume of β-gal at 450 nm was measured in parallel, serving as a control. (B) Cells were treated with LY294002 (20 μM), microcystinLR (25 nM), or LY294002 (20 μM)/microcystin-LR (25 nM) for 24 h. MMP-2/-9 expression was analyzed by Western blotting. (C) Cells were seeded in a 24-well ECM-coated transwell chamber and incubated at 37 °C for 72 h with LY294002 (20 μM) or microcystin-LR (25 nM) or LY294002 (20 μM)/ microcystin-LR (25 nM). The invaded cells were stained with crystal violet and three randomly selected regions were captured under light microscopy ( × 100). One representative experiment of three independent experiments is shown. The OD volume at 570 nm was calculated and analyzed for the stained cells of each sample. *p < 0.05 compared with control cells without microcystin-LR treatment. 8803

dx.doi.org/10.1021/es4007228 | Environ. Sci. Technol. 2013, 47, 8801−8808

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Figure 3. The effect of microcystin-LR on in vivo malignancy of MDA-MB-435 cells in nude mice. (A) Xenograft BALB/c nude mice model. (B) Body weight of mice. (C) Primary tumor size was investigated in mice after cell implantation. (D) Tumor volume and (E) weight. Statistical analyses were carried out by one-way ANOVA.

A p value less than 0.05 was considered statistically significant. Computer-based calculations were conducted using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA).

transcribed using the PrimeScript RT regent kit (Takara, Japan), and qPCR was performed on an ABI 7500 PCR System (Applied Biosystems, ) using Power SYBR Green PCR Master Mix (2X Applied Biosystems, Foster City, CA). Messenger RNA expression of each target gene was determined by normalization to GAPDH. The qPCR primers, reaction systems and conditions recommended by Zhang et al.19 were applied for quantification of mmp2/-9 genes. The qPCR primer set of the internal control gene GAPDH was designed following a previous study.23 IHC. MMP-2/-9 and pAKT protein expression in tumor tissues were assessed by the streptavidin-peroxidase immunohistochemical method using a monoclonal antibody and an UltraSensitive S−P kit (kit 9706, Maixin-Bio, China). Deparaffinized sections were boiled in citrate buffer at a high temperature and high pressure for antigen retrieval, and then incubated with anti-MMP-2 monoclonal antibody (1:100 Cell Signal Technology), anti-MMP-9 monoclonal antibody (1:100, Cell Signal Technology), or pAKT monoclonal antibody (1:100, Epitomics, Burlingame, CA) at 4 °C overnight. Immunohistochemical staining was then performed according to the UltraSensitive S−P kit protocol. Slides were then incubated in diluted diaminobenzidine (Maixin-Bio, China) for 5 min and then washed with ddH2O for 5 min before the slides were counterstained with hematoxylin. Selected regions were captured under light microscopy (×100). Integrated optical density (IOD) of MMP-2, MMP-9 and pAKT protein expression was analyzed by Image Pro Plus (IPP) 5.0 software (Media Cybernetics, Silver Spring, MD). Statistical Analysis. All statistical analyses were carried out by two-tailed Student’s t-test, one-way ANOVA, and the Mann− Whitney test. Data are presented as means ± standard deviation.

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RESULTS

Activation of PI3-K/AKT by Microcystin-LR in MDAMB-435 Cells. In this study, we first investigated the effect of microcystin-LR on protein expression of PI3-K/AKT. MDAMB-435 cells were treated with different concentrations of microcystin-LR (0, 5, 12.5, 25, or 50 nM) and Western blotting demonstrated that microcystin-LR exposure at 25 nM or 50 nM induced phosphorylation of AKT (Figure 1A). Compared with GAPDH expression, PTEN protein expression decreased in a dose-dependent manner with the increase of microcystin-LR dose (≤25 nM) (Figure 1B), which indicated that PI3-K/AKT was activated by microcystin-LR in MDA-MB-435 cells. Role of PI3-K/AKT in Microcystin-LR-Induced MMP-2/-9 Overexpression. We then examined whether microcystin-LR stimulated mRNA and protein expression of MMP-2/-9 via PI3-K/AKT activation in the human melanoma cell line MDAMB-435. MMP-2/-9 expression was investigated at transcriptional level using the mmp-2/-9-binding site-derived luciferase reporter plasmid, suggesting that LY294002 inhibited microcystinLR-induced mmp-2/-9 promoter luciferase activity (Figure 2A). This result was supported by Western blotting assay demonstrating that LY294002 could suppress the MMP-2/-9 protein overexpression induced by microcystin-LR (Figure 2B), which consistently indicated that microcystin-LR stimulated MMP-2/-9 mRNA and protein expression via PI3-K/AKT activation. 8804

dx.doi.org/10.1021/es4007228 | Environ. Sci. Technol. 2013, 47, 8801−8808

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MMP-2, MMP-9, and pAKT Overexpression in MDA-MB435 Tumors. We further assessed the effects of microcystin-LR on MMP-2, MMP-9, and pAKT overexpression in the MDA-MB435 tumors. qPCR assay demonstrated that microcystin-LR upregulated MMP-2/-9 mRNA levels in MDA-MB-435 tumors (Figure 4A). Use of IHC to determine MMP-2/-9 protein expression in formalin-fixed paraffin-embedded MDA-MB-435 tumor samples confirmed that microcystin-LR stimulated MMP-2/-9 expression in the tumors of the nude mice. However, the protein levels of MMP-2/-9 were higher in 0.01 mg/kg/ddose group than in 0.1 mg/kg/d-dose group (Figure 4B and C). Similar to the results of the in vitro experiment, IHC also showed that microcystin-LR treatment at 0.001 or 0.01 mg/kg/ d could up-regulate AKT phosphorylation in the tumors of the nude mice (Figure 4D).

The effects of microcystin-LR on PI3-K/AKT activation were also confirmed by AKT siRNA interference assay (SI Figure S1A). Role of PI3-K/AKT in Microcystin-LR-Induced Cell Invasion. Matrigel chamber assays were conducted to investigate the role of PI3-K/AKT in microcystin-LR-induced cell invasion. After MDA-MB-435 cells were exposed to microcystin-LR (25 nM), more cells were translocated from the above chamber to the lower chamber compared with the control group (Figure 2C), which suggested that microcystinLR could increase the invasive ability. However, the results of chemical suppression assay (Figure 2C) and RNA interference assay (SI Figure S1B) demonstrated that both AKT inhibitor LY294002 and AKT siRNA could suppress the enhancement of invasiveness by microcystin-LR. This finding indicates that PI3K/AKT activation may contribute to the stimulatory effect of microcystin-LR on cell invasion. Effect of Microcystin-LR on Tumor Growth in Nude Mice. An in vivo assay was conducted to evaluate the effect of microcystin-LR on tumor growth by using the MDA-MB-435 xenograft model (Figure 3). BALB/c nude mice were treated with microcystin-LR for 1 week before tumor xenograft. Subsequently, the tumor-xenografted nude mice were orally administrated by microcystin-LR for next 6 weeks. Results showed that the exposure at different doses ranging from 0.001 to 0.1 mg/kg/d posed no evident effect on MDA-MB-435 xenograft tumor size (Figure 3D) and tumor weight (Figure 3E) of the nude mice (p > 0.05), indicating that the microcystin-LR exposure has no significant effect on MDA-MB-435 xenograft tumor size in BALB/c nude mice. Effect of Microcystin-LR on Lung Metastasis in Nude Mice. We then investigated the effects of microcystin-LR on the development of lung metastasis. Microscopic observation showed that lung metastasis occurred after 6 weeks of tumor implantation (SI Figure S2). Histological evaluation demonstrated that lung metastasis took place in two mice in the control group, while the low-, medium-, and high-dose treatment groups had lung metastasis in 4, 3, and 4 mice, respectively. Compared with the control group, the microcystin-LR treatment groups had more areas of lung metastasis (Table 1). In addition, counting the number of tumor cells

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DISCUSSION Current studies mainly focus on the possible mechanisms of microcystin-induced carcinogenesis5,6 or cellular apoptosis,24 but little information is available regarding the microcystin-LR effect on tumor invasion and metastasis. A previous in vitro study has shown that the environmental pollutant microcystinLR can stimulate melanoma cell invasion,17 but whether microcystin-LR has the capacity to promote tumor metastasis in human bodies remains unknown. In the present study, we tested the tumorigenic and metastatic behavior of MDA-MB435 cells in nude mice after microcystin-LR treatment. Oral gavage of microcystin-LR posed no significant effect on the tumor size. This finding is consistent with a previous study indicating that oral administration of mice with microcystins for 154 d cannot affect the tumor formation initiated by N-methylN-nitrosourea.25 In contrast, 52 day exposure to microcystinLR was found to promote tumor growth in mice after treatment with dimethylbenzanthracene, a topical epithelial tumor initiator.26 The different effects of microcystin on tumor growth are likely due to the difference in animal models and tumor types. Notably, this study showed that microcystin-LR exposure stimulated lung metastases in nude mice treated with MDAMB-435 cells by subcutaneous injection, revealing that microcystin-LR has pro-metastatic activity in mice. This hypothesis is supported by the in vitro assay of Matrigel chamber assay demonstrating that exposure to microcystin-LR (25 nM) can accelerate the translocation of MDA-MB-435 cells from the above chamber to the lower chamber. Metastasis is the later progression of cancer, which involves the spread of malignant tumor cells from one organ or part to another nonadjacent organ or part27,28 and they are the major cause of treatment failure in cancer patients. Therefore, our data show that microcystin-LR has the potential ability of promoting malignancy in cancer. Microcystin-LR is frequently detected in eutrophic lakes and reservoirs, many of which are used as source for drinking water in the world.29,30 This causes an increased carcinogenetic risk to people via drinking water3 and food chains.31 Thus, the longterm oral exposure of microcystin-LR to animal models was applied in this study. Due to the relatively low probability that oral administration of microcystin-LR directly causes tumorigenesis in mice,32 previous studies have used various tumor initiators, such as dimethylbenzanthracene,26 diethylnitrosamine,33 N-methyl-N-nitrosourea,25 azoxymethane,34 and aflatoxin B1,35 to improve the cancer incidence before microcystin treatment. However, even when using this method, the incidence of tumor formation is still relatively low, so we

Table 1. Effect of Microcystin-LR on Lung Metastasis of the Tumor-Xenografted Nude Mice with MDA-MB-435 Cella metastases grade groups (mg/kg/d)

number of nude mice (metastases/total)

number of metastases spots

I

II

III

IV

0 0.001 0.01 0.1

2/7 4/7 3/7 4/7

3 14 8 10

2 1 1 1

1 5 1 4

0 3 1 4

0 5 5 3

a

Note: The total number of areas of lung metastasis was counted under a microscope. Metastases were classified into four grades based on the number of tumor cells present in the section for each metastatic lesion: grade I, ≤20 tumor cells; grade II, 21−50 tumor cells; grade III, 51−100 tumor cells; and grade IV, >100 tumor cells. Lung metastases grades were analyzed using the Mann-Whitney test.

present in the tissue sections showed that the grades of lung metastases, especially grades III and IV, were significantly increased after microcystin-LR treatment (Table 1), demonstrating that microcystin-LR exposure increased the possibility of lung metastasis. 8805

dx.doi.org/10.1021/es4007228 | Environ. Sci. Technol. 2013, 47, 8801−8808

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Figure 4. Microcystin-LR-induced MMP-2/-9 and pAKT expression in MDA-MB-435 tumor xenografts. Expression of MMP-2, MMP-9 and pAKT in MDA-MB-435 tumor xenografts was detected by (A) qPCR and (B−D) IHC after the nude mice were exposed to different concentrations of microcystin-LR (0, 0.001, 0.01, and 0.1 mg/kg/d). (A) MMP-2/-9 mRNA levels were determined by qPCR. MMP-2/-9 mRNA levels were normalized to the expression of GAPDH. The differences in MMP-2/-9 mRNA expression were analyzed by one-way ANOVA. Tumors from different groups of mice were used for IHC analysis of (B) MMP-2, (C) MMP-9 and (D) pAKT. Selected regions were captured under light microscopy (×100). The IODs of MMP-2/-9 protein expression were analyzed by IPP software and statistical analyses were carried out by one-way ANOVA. *p < 0.05 and **p < 0.01 denote statistical significance from the control group.

MMP-2/-9 expression and promote cell invasion by accelerating NF-κB translocation into the nucleus and enhancing NF-κB binding ability in MDA-MB-435 cells.17 It is known that PI3-K/ AKT can activate NF-κB via phosphorylation of endogenous inhibitors (IκB) of NF-κB allowing for translocation of the released NF-κB to the nucleus in myeloma cells.39 AKT was also found to stimulate the transactivation potential of the RelA/p65 subunit of NF-κB through activation of the MAPK p38,20 and microcystins can induce constitutive activation of AKT and MAPK.37 Thus, our results demonstrated that microcystin-LR can accelerate NF-κB translocation via PI3-K/ AKT and MAPK activation to enhance MMP-2/-9 expression and promote melanoma cell invasion. No good dose-dependent manner was obtained in this study for cellular effects of microsystins. Previous studies also suggested the contrasting responses to microcystin-LR exposure,40 revealing that low doses of microsystin-LR can increase liver cell survival and proliferation, but higher doses of the toxin can reduce cell viability and induce cell apoptosis. The observed up-regulation of PTEN along with the increase of microcystin-LR concentrations from 25 nM to 50 nM indicated that the melanoma cells began apoptosis under the high stress of microcystin-LR.41 This is the first study revealing that microcystin-LR can promote cancer metastasis based on in vivo evidence. Our previous study has shown that microcystin-LR can stimulate melanoma cell invasion via in vitro assay. It is known that tumor metastasis occurs by a series of steps including invasion, vessel formation, cell attachment, and cell proliferation, and

used nude mice (an athymic mutant strain of mouse) to increase tumorigenesis possibility in this study. Rygaard and Povlsen has indicated that human colon adenocarcinoma grows progressively in nude mice, and this mouse mode has been frequently used in human cancer research.36 To further explore the molecular mechanism underlying the enhancement of cancer cell invasion and metastasis by microcystin-LR, we investigated the role of PI3-K/AKT in MMP-2/-9 overexpression induced by microcystin-LR via in vitro and in vivo exposure. This is the first study revealing that PI3-K/AKT mediates the stimulatory effects of microcystin-LR on MMP-2/-9 expression and tumor metastasis. This result is supported by a previous study indicating that microcystin-LR can activate PI3-K/AKT in cancer cells.9 Previous studies have also shown that microcystin-LR activates AKT in the carcinogenesis of colorectal cancer, indicating that AKT mediates the transformation of immortalized colorectal crypt cells by microcystin-LR.37 In the present study, microcystin-LR treatment caused an increase in pAKT levels in tumor of nude mice, leading to activation of the AKT signaling pathway, which was further confirmed by the PTEN suppression assay. Consistent with the in vivo observation, in vitro exposure of MDA-MB-435 cells to microcystin-LR showed that the PI3-K/ AKT inhibitor LY294002 inhibited the invasiveness increase and MMP-2/-9 overexpression induced by microcystin-LR. AKT siRNA interference assay also revealed the involvement of PI3-K/AKT in the invasiveness increase. PI3-K/AKT controls several cellular responses, including proliferation and survival.38 Our previous study has shown that microcystin-LR can enhance 8806

dx.doi.org/10.1021/es4007228 | Environ. Sci. Technol. 2013, 47, 8801−8808

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oncogenes (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. (7) 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. (8) Komatsu, M.; Furukawa, T.; Ikeda, R.; Takumi, S.; Nong, Q.; Aoyama, K.; Akiyama, S.; Keppler, D.; Takeuchi, T. Involvement of mitogen-activated protein kinase signaling pathways in microcystinLR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3. Toxicol. Sci. 2007, 97, 407−416. (9) 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. (10) 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. (11) Cheng, J. C.; Chou, C. H.; Kuo, M. L.; Hsieh, C. Y. Radiationenhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-kappaB signal transduction pathway. Oncogene 2006, 25, 7009−7018. (12) Zhang, Z.; Song, T.; Jin, Y.; Pan, J.; Zhang, L.; Wang, L.; Li, P. Epidermal growth factor receptor regulates MT1-MMP and MMP-2 synthesis in SiHa cells via both PI3-K/AKT and MAPK/ERK pathways. Int. J. Gynecol Cancer 2009, 19, 998−1003. (13) Burow, M. E.; Weldon, C. B.; Melnik, L. I.; Duong, B. N.; Collins-Burow, B. M.; Beckman, B. S.; McLachlan, J. A. PI3-K/AKT regulation of NF-kappaB signaling events in suppression of TNFinduced apoptosis. Biochem. Biophys. Res. Commun. 2000, 271, 342− 345. (14) Kamada, H.; Nito, C.; Endo, H.; Chan, P. H. Bad as a converging signaling molecule between survival PI3-K/Akt and death JNK in neurons after transient focal cerebral ischemia in rats. J. Cereb Blood Flow Metab. 2007, 27, 521−533. (15) Datta, S. R.; Dudek, H.; Tao, X.; Masters, S.; Fu, H.; Gotoh, Y.; Greenberg, M. E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997, 91, 231−241. (16) Kim, D.; Kim, S.; Koh, H.; Yoon, S. O.; Chung, A. S.; Cho, K. S.; Chung, J. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J. 2001, 15, 1953− 1962. (17) Zhang, X. X.; Fu, Z.; Zhang, Z.; Miao, C.; Xu, P.; Wang, T.; Yang, L.; Cheng, S. Microcystin-LR promotes melanoma cell invasion and enhances matrix metalloproteinase-2/-9 expression mediated by NF-kappaB activation. Environ. Sci. Technol. 2012, 46, 11319−11326. (18) Zhang, Z.; Zhang, X. X.; Qin, W.; Xu, L.; Wang, T.; Cheng, S.; Yang, L. Effects of microcystin-LR exposure on matrix metalloproteinase-2/-9 expression and cancer cell migration. Ecotoxicol. Environ. Saf. 2012, 77, 88−93. (19) Zhang, X. X.; Zhang, Z.; Fu, Z.; Wang, T.; Qin, W.; Xu, L.; Cheng, S.; Yang, L. Stimulation effect of microcystin-LR on matrix metalloproteinase-2/-9 expression in mouse liver. Toxicol. Lett. 2010, 199, 377−382. (20) Madrid, L. V.; Mayo, M. W.; Reuther, J. Y.; Baldwin, A. S., Jr. Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J. Biol. Chem. 2001, 276, 18934−18940. (21) Li, Y.; Tang, Y.; Ye, L.; Liu, B.; Liu, K.; Chen, J.; Xue, Q. Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray. J. Cancer Res. Clin.Oncol. 2003, 129, 43−51. (22) Li, Y.; Tian, B.; Yang, J.; Zhao, L.; Wu, X.; Ye, S. L.; Liu, Y. K.; Tang, Z. Y. Stepwise metastatic human hepatocellular carcinoma cell model system with multiple metastatic potentials established through

invasion is only considered as the initial step in the process of cancer metastasis.42 Thus, this study further indicated that more lung metastasis took place in microcystin-LR-treated nude mice than in the control, confirming that microcystin-LR has the capacity of lung metastasis promotion. This study may extend our knowledge about the effects of microcystin on cancer metastasis. In conclusion, in vivo and in vitro assays show that microcystinLR is able to increase malignancy in cancer by promoting MMP-2/-9 expression and tumor cell invasion. The MMPs overexpression and invasiveness enhancement may be mediated by activation of PI3-K/AKT to accelerate NF-κB translocation. This is the first study revealing that PI3-K/AKT mediates the stimulation of microcystin-LR in MMP-2/-9 expression and tumor metastasis. However, the underlying mechanism behind the activation of PI3-K/AKT by microcystin-LR still needs indepth identification. During cyanobacterial bloom, microcystinLR is produced in many lakes and reservoirs that often serve as source of drinking water for local residents. Therefore, our findings indicate the importance of assessing the new public health risks (e.g., tumor metastasis promotion) induced by this toxin in drinking water.

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ASSOCIATED CONTENT

S Supporting Information *

The role of AKT in microcystin-LR-enhanced cell invasive ability (Figure S1), and MDA-MB-435 metastasis in the lungs of nude mice (Figure S2). 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 ⊥

P.X., X.X.Z.: 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 Natural Science Foundation of China (21177062) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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REFERENCES

(1) Nishizawa, T.; Asayama, M.; Fujii, K.; Harada, K.; Shirai, M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J Biochem 1999, 126, 520−529. (2) Martinez Hernandez, J.; 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. (3) Zhou, L.; Yu, H.; Chen, K. Relationship between microcystin in drinking water and colorectal cancer. Biomed Environ Sci 2002, 15, 166−171. (4) Zegura, B.; Straser, A.; Filipic, M. Genotoxicity and potential carcinogenicity of cyanobacterial toxins - a review. Mutat. Res. 2011, 727, 16−41. (5) Zegura, B.; Zajc, I.; Lah, T. T.; Filipic, M. Patterns of microcystinLR induced alteration of the expression of genes involved in response to DNA damage and apoptosis. Toxicon 2008, 51, 615−623. (6) Li, H.; Xie, P.; Li, G.; Hao, L.; Xiong, Q. In vivo study on the effects of microcystin extracts on the expression profiles of proto8807

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Environmental Science & Technology

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consecutive in vivo selection and studies on metastatic characteristics. J. Cancer Res. Clin. Oncol. 2004, 130, 460−468. (23) Li, G.; Chen, J.; Xie, P.; Jiang, Y.; Wu, L.; Zhang, X. Protein expression profiling in the zebrafish (Danio rerio) embryos exposed to the microcystin-LR. Proteomics 2011, 11, 2003−2018. (24) Ji, Y.; Lu, G.; Chen, G.; Huang, B.; Zhang, X.; Shen, K.; Wu, S. Microcystin-LR induces apoptosis via NF-kappaB/iNOS pathway in INS-1 cells. Int. J. Mol. Sci. 2011, 12, 4722−4734. (25) Falconer, I. R.; Humpage, A. R. Tumour promotion by cyanobacterial toxins. Phycologia 1996, 35, 74−79. (26) Falconer, I. R. Tumor promotion and liver injury caused by oral consumption of cyanobacteria. Environ Toxicol Water Qual 1991, 6, 177−184. (27) Steeg, P. S. Tumor metastasis: Mechanistic insights and clinical challenges. Nat. Med. 2006, 12, 895−904. (28) Nguyen, D. X.; Massague, J. Genetic determinants of cancer metastasis. Nat. Rev. Genet. 2007, 8, 341−352. (29) Wood, S.; Holland, P.; Stirling, D.; Briggs, L.; Sprosen, J.; Ruck, J.; Wear, R. Survey of cyanotoxins in New Zealand water bodies between 2001 and 2004. N. Z. J. Mar. Freshwater Res. 2006, 40, 585− 597. (30) Otten, T. G.; Xu, H.; Qin, B.; Zhu, G.; Paerl, H. W. Spatiotemporal patterns and ecophysiology of toxigenic microcystis blooms in Lake Taihu, China: Implications for water quality management. Environ. Sci. Technol. 2012, 46, 3480−3488. (31) Anderson, D.; White, A. Marine biotoxins at the top of the food chain. Oceanus 1992, 35, 55−61. (32) Ito, E.; Kondo, F.; Terao, K.; Harada, K. Neoplastic nodular formation in mouse liver induced by repeated intraperitoneal injections of microcystin-LR. Toxicon 1997, 35, 1453−1457. (33) Nishiwaki-Matsushima, R.; Ohta, T.; Nishiwaki, S.; Suganuma, M.; Kohyama, K.; Ishikawa, T.; Carmichael, W. W.; Fujiki, H. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol. 1992, 118, 420−424. (34) Humpage, A. R.; Hardy, S. J.; Moore, E. J.; Froscio, S. M.; Falconer, I. R. Microcystins (cyanobacterial toxins) in drinking water enhance the growth of aberrant crypt foci in the mouse colon. J .Toxicol. Environ. Health A 2000, 61, 155−165. (35) Sekijima, M.; Tsutsumi, T.; Yoshida, T.; Harada, T.; Tashiro, F.; Chen, G.; Yu, S. Z.; Ueno, Y. Enhancement of glutathione Stransferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats. Carcinogenesis 1999, 20, 161−165. (36) Rygaard, J.; Poulsen, C. O. Heterotransplantation of a human malignant tumour to “Nude” mice. Acta Pathol. Microbiol. Scand. 1969, 77, 758−760. (37) Zhu, Y.; Zhong, X.; Zheng, S.; Ge, Z.; Du, Q.; Zhang, S. Transformation of immortalized colorectal crypt cells by microcystin involving constitutive activation of Akt and MAPK cascade. Carcinogenesis 2005, 26, 1207−1214. (38) Tu, Y.; Gardner, A.; Lichtenstein, A. The phosphatidylinositol 3kinase/AKT kinase pathway in multiple myeloma plasma cells: Roles in cytokine-dependent survival and proliferative responses. Cancer Res. 2000, 60, 6763−6770. (39) Mitsiades, C. S.; Mitsiades, N.; Poulaki, V.; Schlossman, R.; Akiyama, M.; Chauhan, D.; Hideshima, T.; Treon, S. P.; Munshi, N. C.; Richardson, P. G.; Anderson, K. C. Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: Therapeutic implications. Oncogene 2002, 21, 5673−5683. (40) Gehringer, M. M. Microcystin-LR and okadaic acid-induced cellular effects: A dualistic response. FEBS Lett. 2004, 557, 1−8. (41) Chen, Z.; Trotman, L. C.; Shaffer, D.; Lin, H. K.; Dotan, Z. A.; Niki, M.; Koutcher, J. A.; Scher, H. I.; Ludwig, T.; Gerald, W. Crucial role of p53-dependent cellular senescence in suppression of Ptendeficient tumorigenesis. Nature 2005, 436, 725−30. (42) Chaffer, C. L.; Weinberg, R. A. A perspective on cancer cell metastasis. Science 2011, 331, 1559−1564.

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