Fucoidan from Seaweed Fucus vesiculosus Inhibits Migration and Invasion of Human Lung Cancer Cell via PI3K-Akt-mTOR Pathways

Background Recently there has been an increased interest in the pharmacologically active natural products associated with remedies of various kinds of diseases, including cancer. Fucoidan is a polysaccharide derived from brown seaweeds and has long been used as an ingredient in some dietary supplement products. Although fucoidan has been known to have anti-cancer activity, the anti-metastatic effects and its detailed mechanism of actions have been poorly understood. Therefore, the aims of this study were to demonstrate the anti-metastatic functions of fucoidan and its mechanism of action using A549, a highly metastatic human lung cancer cell line. Methods and Principal Findings Fucoidan inhibits the growth of A549 cells at the concentration of 400 µg/ml. Fucoidan treatment of non-toxic dose (0–200 µg/ml) exhibits a concentration-dependent inhibitory effect on the invasion and migration of the cancer cell via decreasing its MMP-2 activity. To know the mechanism of these inhibitory effects, Western blotting was performed. Fucoidan treatment down-regulates extracellular signal-related kinase 1 and 2 (ERK1/2) and phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (PI3K-Akt-mTOR) pathways. Furthermore, fucoidan decreases the cytosolic and nuclear levels of Nuclear Factor-kappa B (p65). Conclusions/Significance The present study suggests that fucoidan exhibits anti-metastatic effect on A549 lung cancer cells via the down-regulation of ERK1/2 and Akt-mTOR as well as NF-kB signaling pathways. Hence, fucoidan can be considered as a potential therapeutic reagent against the metastasis of invasive human lung cancer cells.


Introduction
Metastasis is a leading cause (up to 90%) of cancer-related deaths. The development of cancer metastasis consists of multiple processes, in which cancer cells first detach from the primary tumor, invade surrounding tissues and intravasate into blood and/ or lymphatic systems and extravasate from the vasculature and subsequently settle and colonize at the target organs. Matrix metalloproteinases (MMPs) play a key role in tumor metastasis, where MMPs degrade extracellular matrix (ECM) proteins such as collagen, proteoglycan, elastin, laminin, and fibronectin [1,2]. Among them, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are expressed abundantly in various malignant cancers and degrade type IV collagen, which is a major component of the basement membrane. Generally, MMP-2 is over-expressed constitutively in highly metastatic cancers, whereas MMP-9 is induced by some stimulating factors such as inflammatory cytokines, epidermal growth factor and phorbol-12-myristate-13acetate [3]. Therefore, MMP-2 may play a more important role in the cancer cells migration and invasion.
Lung cancer is one of the most common cancers in the world and a leading cause of cancer death, accounting for more than a million deaths yearly worldwide [4]. In particular, it is an extremely aggressive and metastatic tumor probably due to secreting higher levels of MMPs comparing with other cancers. Hence, the inhibition of MMP-2 in cancer cells appears to be an interesting therapeutic target of highly metastatic lung cancer cells. MMPs expression is regulated by transcriptional factors (AP-1 and NF-kB) through upstream pathways including mitogen activated kinases (MAPKs) and PI3K-Akt pathways [5]. MAPKs consist of three kinases, including extracellular signal-related kinase 1 and 2 (ERK1/2), c-JUN N-terminal kinase/stress activated protein kinase (JNK/SAPK), and p38. MAPKs are implicated in a variety of cellular functions such as proliferation, migration and invasion [3]. Activation of PI3K can stimulate its downstream target, Akt, and then regulate cell growth, adhesion, and invasion [6].
There have been numerous reports investigating the cancer chemopreventive or therapeutic agents from natural products. Many species of marine algae have long been used in food diet and also documented as being used in traditional oriental medicine for over 1000 years [7,8]. They contain various functional compo-nents, such as porphyran, gepsin, alginic acid, and oligosaccharide. Fucoidan, whose molecular weight average is about 20,000, is a sulphated polysaccharide extracted from brown marine algae and consists of L-fucose and sulphate ester groups. It has been previously suggested that fucoidan has various biological activities such as antibacterial [9], antioxidant [10], anti-inflammatory [11], anticoagulant [12], and anti-tumor activities [2]. In C57BL/6 mice transplanted with Lewis lung adenocarcinoma cells, fucoidan from Fucus evanescens produced moderate antitumor and antimetastatic effects and potentiated the anti-metastatic, but not antitumor activities of cyclophosphamide [13]. In addition, fucoidan inhibited MMP-2/9 activities and invasiveness of HT-1080 cells and formation of vascular tubules via suppressing expression and secretion of an angiogenesis factor [14]. However, molecular mechanism of its action has been rarely understood yet.
In the present study, it was investigated the effect of fucoidan on migration, invasion and MMP-2 expression of A549 human lung cancer cells and its underlying anti-metastatic mechanisms of action.

Cell Culture
A549 cells from ATCC (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 supplemented with 10% FBS, 100 mg/ml penicillin-streptomycin-amphotericin B solution at 37 uC in a 5% CO 2 humidified incubator. Cells were passaged three times a week by treating with trypsin-EDTA and those of after five passages were used for experiments.

MTT Assay for Cell Viability
Cell viability was measured by MTT (3-(4,5-dimethyl-2-yl)-2,5diphenyltetrazolium bromide) reduction assay as described previously [15]. Briefly, A549 cells were incubated at a density of 4610 4 cells/well in 24-well plates for 24 h. The cells were treated with various concentrations of fucoidan for 48 h. Then, MTT dye (5 mg/ml) was added to the cells and they were incubated for additional 3 h. After the medium was removed, DMSO was added to the cells for the solubilization of generated formazan salts. The amount of formazan salt was determined by measuring the optical density (OD) at 540 nm using GENiosH microplate spectrophotometer (PowerWave TM XS, BioTek Instruments, Inc., Winooski, USA). Relative cell viability of treatment was calculated as a percentage of vehicle-treated control ([OD of treated cells -OD of blank/OD of control -OD of blank] 6 100).

Wound Migration Assay
Cell migration assay was performed using 12-well plates as previously described [16]. A549 cells were seeded into 12-well plates (1610 5 cells/ml) and grown to 80,90% confluence for the experiment. After aspirating the medium, cells were scraped with a 200 ml sterile pipette tip to create a straight scratch. They were washed twice with PBS to remove cellular debris and then replaced with complete RPMI 1640. A549 cells were treated with fucoidan (0, 50, 100, and 200 mg/ml) and incubated for 48 hr. Cell migration into the wound area was photographed at the stages of 0 hr and 48 hr, respectively, for the image analysis of each treatment. The level of cell migration was determined using a Hewlett-Packard scanner and NIH Image software (Image J), and then it was expressed as a percentage of each control for the mean of wound closure area.

Cell Invasion Assay
The invasive behavior of A549 cells was tested using cell invasion chamber kit (BD Bioscience, San Jose, CA, USA) [17]. A549 cells were resuspended in a serum-free RPMI 1640 medium (5610 4 cells/200 ml) in the absence or the presence of fucoidan (0, 50, 100 and 200 mg/ml). The cells were seeded onto the upper chamber of Matrigel-coated filter and a RPMI 1640 containing 10% FBS was added 500 ml in the lower chamber. After 48 hr incubation, the non-invading cells were removed from the upper surface of the filter membrane. The invading cells on the lower surface of the filter membrane were stained with crystal violet for 1 hr and rinsed with water and dried. The amount of invading cells on the lower surface of filter membrane was determined using a light microscope and NIH Image software (Image J).

Preparation of Cell Lysates
After fucoidan treatments, A549 cells were rinsed twice with icecold PBS, and then added with 100 mL of lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM Na 3 VO 4 , 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin). The lysis reaction was performed on ice with rocking for 3 min and the cells were scraped using rubber policeman into Eppendorf microcentrifuge tubes. The scraped cells were allowed to lyse for additional 30 min on ice with periodic vortexing. Cell debris was removed by centrifugation (22,0006g, at 4uC for 30 min) and the resulting supernatant was collected and determined for its protein concentration using a Bio-Rad protein assay reagent (Bio-Rad, C.A. USA). Samples were cooked with SDS sample buffer in boiling water for 5 min.

Western Blotting
The protein components (50 mg) were separated on 12% SDSpolyacrylamide gel, transferred to polyvinylidene difluoride membranes (Bio-Rad, C.A. USA), and subsequently subjected to immunoblot analysis using specific primary antibodies for overnight at 4uC. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA) for 1 hr at room temperature. The blots were then visualized by using the enhanced chemiluminescence method (ECL, Amersham Biosciences, Buckinghamshire, UK) on blue light-sensitive film (Fujifilm Corporation, Tokyo, Japan). If necessary, the blotted membranes were stripped and reprobed using other primary antibodies. Densitometry analysis was performed with a Hewlett-Packard scanner and NIH Image software (Image J).

Statistical Analysis
The results are expressed as the mean 6 standard deviation (S.D.). One-way analysis of variance (ANOVA) was used to evaluate the significance of difference between the two mean values. *P,0.05 and **P,0.01 were considered to be statistically significant.

Fucoidan Inhibits the Activity of MMP-2 in A549 Human Lung Cancer Cells
In order to determine non-cytotoxic concentration of fucoidan, A549 cells were incubated with various concentrations of fucoidan (0-1000 mg/ml) for 24 hr or 48 hr, and then cell viability was determined using MTT assay (data not shown). Fucoidan did not show any significant toxic effect on A549 cells within the concentration range of 0-200 mg/ml. At higher concentrations (400-1000 mg/ml), however, fucoidan inhibited cell proliferation and decreased cell viability in a concentration-dependent manner. Therefore, the present study applied the treatment of fucoidan at concentrations equal to or lower than 200 mg/ml, at which no significant cytotoxic effect of A549 cells was observed.
The degradation of extracellular matrix (ECM) is crucial for cellular invasion, indicating the inevitable involvement of matrixdegrading proteinases for the process. Hence, it was assessed the effect of fucoidan on MMP-2 activity, as a key molecule of ECM degradation, by gelatin zymography. As shown in Figure 1A, fucoidan suppressed MMP-2 activity in a concentration-dependent manner. On the other hand, the impact of fucoidan on the MMP-9 activity was inconclusive, since there is only an extremely low level of intrinsic MMP-9 activity detectable in A549 cells (data not shown). MMP-2 activity was reduced by 36%, 53%, and 72% at 50, 100, and 200 mg/ml, respectively, after treatment with the  fucoidan for 48 hr (Fig. 1B). The results obtained by zymography were further confirmed by Western blot analysis. MMP-2 expression was gradually decreased with increasing concentrations of fucoidan (Fig. 1C). The protein level was reduced by 38%, 48%, and 60% at 50, 100, and 200 mg/ml, respectively. The reduction of MMP-2 activity was probably due to the decrease in MMP-2 expression (Fig. 1B and 1D).

Fucoidan Suppresses the Migration of A549 Cells
Cell migration is a measure of metastatic potential of cancer cells. Effects of fucoidan on cell migration were investigated using a wound healing assay. A549 cells treated with fucoidan showed reduction of migration ( Fig. 2A). As shown in Figure 2B, cell migration was inhibited by up to 25%, 46%, and 57% at 48 hr in the presence of 50, 100, and 200 mg/ml of fucoidan, respectively. Collectively, these data demonstrated that fucoidan may suppress the migratory property of lung cancer cells.

Fucoidan Inhibits the Invasion of A549 Cells
To elucidate inhibitory effect of fucoidan on invasion of A549 cells, it was tested using invasion chamber kit (BD Bioscience, San Jose, CA, USA), in which cells invade across extracellular matrix, such as Matrigel-coated filter in chamber. As illustrated in Figure 3A, the number of cells with invasion (crystal-violet staining) reduced remarkably in a concentration-dependent manner, suggesting fucoidan can inhibit the invasion of A549 cells. The inhibitory effects of fucoidan on cancer cell invasion were 35%, 68% and 86% at the concentrations of 50, 100, and 200 mg/ml of fucoidan, respectively (Fig. 3B). These results indicated that fucoidan can directly inhibit the invasive potential of lung cancer cell.

Fucoidan Inhibits the MMP-2 Activity by Blocking ERK1/2 and PI3K-Akt Pathways in A549 Cells
Our findings showed that fucoidan dramatically inhibits migration, invasion and MMP-2 activity in A549 cells. However, the signal mechanisms responsible for the inhibitory effect of fucoidan on metastasis remain to be elucidated. Several evidences suggest that MAPKs (JNK 1/2, ERK 1/2, and p38) play important roles in cancer cell migration, invasion, and MMP-2 activity [5]. Considering the strong anti-metastatic potential of fucoidan, it was tested whether it can regulate the signal  Figure 4 showed that fucoidan reduced phosphorylation of ERK1/2 in a concentration-and time-dependent manner, whereas it had almost no or marginal, if any, effect on p38 and JNK1/2. As shown in Figure 4A and 4C, a time-dependent study demonstrated that fucoidan progressively reduced phosphorylation of ERK1/2 from 1 hr to 9 hr after the treatment. Figure 4B and 4D showed that fucoidan significantly inhibited phospho-ERK1/2 in a concentration-dependent manner with a maximum inhibitory effect at the highest concentration (200 mg/ml). In addition to MAPKs, the cell signaling of PI3K/Akt has been also proposed as a key player in the regulation of MMP-2 activity [19]. Our current study revealed that fucoidan markedly inhibited the phosphorylations of PI3K and its downstream target, Akt, in a concentration-and time-dependent manner (Fig. 5). Based on these results, fucoidan potently inhibited the metastatic activity of human lung cancer cells, possibly by the suppressions of ERK1/2 and PI3K-Akt pathways.

Fucoidan Suppresses mTOR Signaling
It has been reported that PI3K is associated with a number of cellular processes including proliferation, differentiation, apoptosis, cell motility, invasion and angiogenesis [20,21]. The signaling of mTOR has been emerged especially as a critical effecter of PI3K signal transduction pathway in various human cancers [22]. Based on our finding that fucoidan suppresses PI3K-Akt pathway in human lung cancer cells, it was examined whether fucoidan modulates mTOR and its downstream signaling molecules. As shown in Figure 6A and 6B, fucoidan inhibited the phosphorylation of mTOR in a concentration-dependent manner. Additionally, 4E-BP1 and p70S6K, two immediate downstream targets of mTOR and indicators of mTOR activity, were also significantly suppressed ( Fig. 6C and 6D).

Fucoidan Inhibits MMP-2 Activity by Blocking ERK1/2 and PI3K-Akt-mTOR Pathways in A549 Cells
To confirm whether the inhibition of MMP-2 activity by fucoidan mainly rely on its suppression of ERK1/2 and PI3K-Akt-mTOR pathways, A549 cells were pretreated in the presence or absence of U0126 (an ERK inhibitor), LY294002 (a PI3K inhibitor) or rapamycin (a mTOR inhibitor) for 2 hr, then examined for the effects of fucoidan treatment (Fig. 7). Compared with the respective vehicle controls, individual treatments with U0126 (Fig. 7A, lane 3 and 4), LY294002 (Fig. 7B, lane 3 and 4) or rapamycin (Fig. 7C, lane 3 and 4) gave a significant reduction of MMP-2 activity. The results are consistent with those of fucoidan experiments of the present study in that MMP-2 inhibition can be mediated by the modulations of ERK1/2, PI3K-Akt and/or mTOR pathways. Describing in detail, the cotreatment of U0126 (10 mM and 20 mM) and fucoidan further reduced the MMP-2 activity by 66% and 75% compared with U0126 alone (46% and 65%) (Fig. 7A). In the same manner, the combined treatments of LY294002 and fucoidan additionally suppressed the MMP-2 activity by 65% and 86% in comparison with LY294002 alone (27% and 43%) (Fig. 7B). However, unlike others, rapamycin and fucoidan cotreatment did not give an additional decrease of MMP-2 activity compared with rapamycin alone (Fig. 7C). It is not yet clearly understood why there was neither additive nor synergistic effects of rapamycin with fucoidan. In summary, the inhibition of MMP2 by fucoidan treatment is possibly exhibited by the modulations of ERK1/2 and/or PI3K-Akt-mTOR pathways. These results proposed the therapeutic potential of fucoidan as an anti-metastatic agent in human lung cancer patients.

Fucoidan Reduces the Nuclear Translocation of NF-kB
NF-kB pathway has been demonstrated to rely upon sequentially activated kinase cascades [23]. On activation, NF-kB translocates from cytoplasm into nucleus and regulates the expression of a wide variety of target genes, including MMPs [5]. Since NF-kB can play a key role in MMP-2 gene expression, the effects of fucoidan was assessed on IkBa and NF-kB signal transduction. As shown in Figure 8A and 8B, fucoidan inhibited the phosphorylation of IkBa in a concentration-dependent manner, whereas the total IkBa was inversely increased by fucoidan. Being consistent with this, phospho-p65, an indicator of NF-kB activation through IkBa degradation, was decreased by the treatment of fucoidan (Fig. 8A and 8C), which is accompanied by the increase of total p65 in the cytosolic fraction and its decrease in nuclear fraction (Fig. 8A and 8D). The results demonstrated that fucoidan significantly inhibited activation of NF-kB in A549 cells.

Discussion
It is well known that MMPs provide an access for tumor cells to the vascular and lymphatic vessels, which support tumor growth and constitute an escape route for further dissemination [24]. Furthermore, MMPs promote malignant transformation in early stages of cancer and suppress cancer cell apoptosis and enhance angiogenesis as well as destroy chemokines balance by host antitumor defense system [25]. Accordingly, blocking of MMPs activity and expression may lead to the development of effective therapy in metastatic cancer patients. Although many scientists have made great efforts to develop potential anticancer agents of MMPs inhibitors, most clinical trials have not been successfully completed yet [26].
In search of efficacious metastatic suppressors, a wide variety of natural products and their pharmacological ingredients can be a good source of finding promising candidates, having several advantages over synthetic chemicals on side effects and pharmacophore diversity. Some of them from various natural resources have been already characterized as potential therapeutics with anti-metastatic activity [27]. Among them, fucoidan has been revealed to possess anti-proliferative and cytotoxic effects on MCF-7 breast cancer cells, but not human mammary epithelial cells (HMECs) [28]. Recently, fucoidan has been demonstrated to inhibit metastasis via MMP-2/29 suppression as well as subdue the expression and secretion of a vascular endothelial growth factor (VEGF) [14]. The anti-metastatic activity of fucoidan was also proven in the animal model of experimental transplanted Lewis lung carcinoma (LLC) [13]. However, the inhibitory mechanism on cancer metastasis has not been clearly elucidated in detail yet. For the first time, in this study, it is at least partly identified the anti-metastatic molecular mechanism of fucoidan.
The status of cell viability was assessed using MTT assay in the absence or presence of various concentrations of fucoidan. Fucoidan inhibited cell growth at 400 mg/ml but not at between 0 to 200 mg/ml, at which it effectively inhibited migration and invasion without significant cytotoxic effect. Therefore, the antiinvasive and anti-migratory effects of fucoidan observed from the present study were not dependent on its anti-proliferation effect. The enhancement of MMP-2 activity has been well characterized in highly metastatic human lung cancer cells [29]. However, MMP-9 is still inconclusive, since there is only an extremely low level of MMP-9 detected in A549 human lung cancer cells. Based on our present study, the anti-metastatic effects of fucoidan appear to be mediated by its inhibitory activity on MMP-2. (Figs. 1, 2 and  3). Our results show that fucoidan effect is more like the suppression of MMP-2 secretion and/or expression rather than the direct inhibition of its activity. ERK1/2 pathway is involved in the invasive or migratory behavior of a number of malignancies, such as colon cancer, melanoma, breast cancer, and prostate cancer and this is wellestablished in previous studies [30][31][32]. Moreover, ERK1/2 regulates focal adhesion and cytoskeletal reorganization via the phosphorylations of specific cytoskeletal and focal adhesion proteins, including paxillin, FAK, myosin light chain kinase [33]. As shown in Figure 4, the anti-metastatic effect of fucoidan is owing to its inactivation of ERK1/2 pathway in A549 human lung cancer cells. In addition to ERK1/2, the PI3K-Akt signal pathway has been shown to regulate the invasion and metastasis of nonsmall-cell lung cancer (NSCLC) as well as the development and progress of various other tumors. That is to say, PI3K overexpression is highly correlated with the development, invasion, and metastasis of NSCLC [34]. Several other studies have also shown that targeting of PI3K-Akt signaling pathway with antisense, siRNA or small molecule inhibitors results in the down-regulation of tumor invasion and tumorigenesis in malignant cancer cells [6,35]. The results of Figure 5 exhibited that fucoidan inhibits the phosphorylation of PI3K-Akt in time-and concentration-dependent manners.
Mammalian target of Rapamycin (mTOR) is a serine/threonine protein kinase that regulates cell growth and proliferation, cell motility, cell survival, protein synthesis, and transcription [36,37]. The dysregulation of mTOR pathway can be observed in human diseases, especially certain cancers, and there are also some mTOR inhibitors, which are beginning to be used in the treatment of cancer [22]. PI3K and Akt are well known upstream regulators of mTOR signaling pathway in mammalian cells. As an evidence of this notion, siRNA-mediated gene silencing of PI3K and Akt inhibited the activation of p70S6K1 (a downstream target of mTOR), and subsequently led to the suppression of migration, invasion and proliferation [38]. It has also been reported that mTOR pathway regulates MMP-2 and -9 expressions and angiogenesis through modulating VEGF gene expression. The treatment of rapamycin (a mTOR inhibitor) and siRNA-mediated mTOR knockdown inhibit tumor cell invasion as well as the secretions of MMP-2 and uPA [21,39]. As shown in Figure 6, fucoidan treatment significantly decreased the phosphorylations of mTOR and its downstream target molecules, such as p70S6K1 and 4EBP1. The present study demonstrated that fucoidan effectively down regulates the expression of MMP-2 through the inhibitions of PI3K-Akt-mTOR as well as ERK1/2 signaling pathways in A549 human lung cancer cells. To further support these findings, MMP-2 activity was examined upon the cotreatments of fucoidan with ERK1/2, PI3K or mTOR inhibitor. The MMP-2 suppression induced by the cotreatment is more likely synergistic than additive effect, except mTOR inhibitor. (Fig. 7). Interestingly, unlike other ones, rapamycin co-treatment with fucoidan showed neither synergistic nor additive effect, which is not yet clearly understood. One of the possible interpretations might be the intrinsic limit of efficacy of mTOR signaling pathway in the regulation of MMP2 activity.
NF-kB and AP-1 are transcription factors that regulate the expressions of numerous genes associated with many important biological and pathological processes, including cancer. It has been reported that the Inhibition of NF-kB and AP-1 results in the suppression of tumor initiation, promotion and metastasis [33,40]. MMP gene expressions are also regulated by NF-kB and AP-1, hence the blocking of these transcription factors is likely to be a promising strategy for cancer therapy aiming at the down regulation of MMP genes [41]. As shown in Figure 8, fucoidan treatment prohibited the degradation of I k Ba protein and the translocation of NF-kB p65 to the nucleus.
In conclusion, it is the first time that fucoidan is revealed to inhibit MMP-2 activity via the suppression of PI3K-Akt-mTOR and NF-kB signaling pathway in A549 lung cancer cells, resulting in the down regulation of cancer cell migration and invasion. Further in vivo study and clinical investigations are necessary for the application of fucoidan as a novel therapeutic agent and alternative remedy in metastatic cancer patients. By any measure, Figure 8. Effect of fucoidan on NF-kB activation in A549 cells. A549 cells were incubated for 24 hr with fucoidan at various concentrations (0, 50, 100 and 200 mg/ml). (A) The cytosolic extract were prepared and analyzed by Western blotting with phospho-IkB and phospho-and total-NF-kB (p65). (B, C and D) The level of phospho-IkB and phosphor-and total NF-kB were quantified by the analysis with Image J software. GAPDH was used as a loading control in cytosolic fraction and Lamin B was loading control in nuclear fraction. The band intensities of each signaling molecules were quantified by the analysis with Image J software. The data shown are the means 6 SD of three experiments. Significant difference from control group, *p,0.05 and **p,0.01. doi:10.1371/journal.pone.0050624.g008 fucoidan looks promising as an efficacious anti-metastatic reagent with its low side effect in normal cells.