Food Chemistry 127 (2011) 1229–1236

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Antimetastatic activity of polyphenol-rich extract of Ecklonia cava through the inhibition of the Akt pathway in A549 human lung cancer cells Hyunkyoung Lee a, Changkeun Kang a, Eun-sun Jung a, Jong-Shu Kim a, Euikyung Kim a,b,⇑ a b

Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, South Korea Research Institute of Life Science, Gyeongsang National University, South Korea

a r t i c l e

i n f o

Article history: Received 18 August 2010 Received in revised form 26 December 2010 Accepted 1 February 2011 Available online 24 February 2011 Keywords: Ecklonia cava Cancer cell Invasion MMP-2 Akt p38

a b s t r a c t An ethyl acetate extract (ECE) of a brown alga, Ecklonia cava, was examined for its anti-metastatic effect, using A549 human lung carcinoma cells. ECE treatment significantly suppressed the migration and invasion of A549 cells in a concentration-dependent manner. It also strongly down-regulated the matrix metalloproteinase (MMP)-2 activity of the cancer cells by gelatin zymography assay. For elucidating its mechanism of action in cancer cell metastasis, ECE was further investigated for various cell signalling pathways, including JNK, ERK, p38, and Akt. In this, ECE showed an anti-metastatic effect in a concentration- and time-dependent manner by the mechanism of suppression of Akt and p38, but not JNK and ERK. These results, for the first time, suggest that ECE (a polyphenol-enriched, highly anti-oxidative fraction of brown alga, E. cava) may have therapeutic potential in metastatic lung cancer, based on its strong inhibitory effects on the migration and invasiveness of A549 human lung adenocarcinoma cells. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Lung cancer is now the most common cause of cancer-related deaths, which accounts for more than one million worldwide annual deaths (Jemal et al., 2008). It is one of the most malignant types of human cancer and about 70% of patients die as a result of cancer metastasis. Approximately, 80% of people diagnosed with lung cancer have non-small cell lung cancer (NSCLC), such as adenocarcinoma, which remains an aggressive cancer with poor prognosis. Therefore, our knowledge on the invasion and migration of lung cancer cells is crucial for designing a new therapeutic strategy against the malignant lung tumour. In cancer cell metastasis, the degradation of extracellular matrix (ECM) is essential; this is associated with the overexpression of proteolytic enzymes, such as matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). MMP-2 and MMP-9 can degrade most of the ECM components of basal membrane and the strong expression of MMP-2 has been well characterised in highly metastatic human lung cancer cells, such as A549 cell (Chu, Chiou, Chen, Yang, & Hsieh, 2004). Up to now, the search for MMP-2 inhibitors has been a focus of interest for the development of anti-metastatic agents. However, currently available inhibitors have often exhibited severe side effects at their thera⇑ Corresponding author at: Department of Pharmacology and Toxicology, College of Veterinary Medicine, Gyeongsang National University, 900 Gajwa-dong, Jinju 660-701, South Korea. Tel.: +82 55 751 5812; fax: +82 55 751 5803. E-mail address: [email protected] (E. Kim). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.005

peutic doses, including inflammation, musculoskeletal pain and joint stiffness (Whittaker, Floyd, Brown, & Gearing, 1999). Therefore, it is important to develop a novel MMP inhibitor with fewer side effects and a high therapeutic efficacy. It has been achieved by library screenings of various origins such as natural products. For example, curcumin (turmeric) has been observed to block invasion and migration of malignant tumour cells (Duvoix et al., 2005) and it inhibits metastasis in animal models (Menon, Kuttan, & Kuttan, 1999). In murine melanoma cells, the treatment with curcumin reduced the activities of MMP-2 and MT1-MMP (Banerji, Chakrabarti, Mitra, & Chatterjee, 2004). In general, seaweeds are a rich source of natural antioxidants and some of them are known as pigments (e.g. fucoxanthin, astaxanthin, and carotenoids) and polyphenols (phenolic acid, flavonoid, and tannins) (Heo, Park, Lee, & Jeon, 2005). Among these components, phlorotannins, a class of compounds with polymerised phloroglucinol units found in brown algae, especially in Ecklonia cava (E. cava), have strong antioxidant activities. E. cava is a member of the family of Laminariaceae, which belongs to the order Laminariales, as a perennial brown alga. It is distributed prolifically along the coasts of South Korea and Japan. It has long been used in Korea, in food ingredients, cosmetics and folk medicine in gynecopathy (Ahn et al., 2007). There are several phloroglucinol derivatives in E. cava, and its crude methanol extract, and single phlorotannins have proved to have various biological activities, including radical-scavenging (Kang et al., 2004), and anti-allergic (Kim et al., 2008), bactericidal and protease inhibition effects (Ahn et al., 2004). However, the activity of E. cava on cancer cell

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metastasis remains poorly understood. In the present study, we have elucidated the anti-metastatic potential of the ethyl acetate fraction of E. cava (ECE) and its regulatory mechanism, using A549 human lung carcinoma cells. 2. Materials and methods 2.1. Chemicals and reagents RPMI 1640 medium without phenol red, Trypsin–EDTA, penicillin–streptomycin–amphotericin B solution, fetal bovine serum (FBS), and phosphate buffer solution (PBS) were from Gibco BRL, Life Technologies (USA). MTT reagent, gelatin (G8150), Folin–Ciocalteu’s phenol reagent, and gallic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). LY294002 (a PI3K inhibitor) was from Tocris Cookson Ltd. (Bristol, UK). For ERK1/2, p38, JNK, and Akt, their total and/or phosphorylated protein-specific antibodies were purchased from Cell Signaling Technology (Beverly, MA). Invasion assay chambers ware obtained from BD Biosciences (San Jose, CA, USA). 2.2. Plant material and extraction E. cava, a brown seaweed, was collected along the coast of Jeju island, South Korea, during the month of March. Fresh E. cava was washed three times with tap water to remove salt and impurities, then dried at room temperature for 3 days and stored at 20 °C. The dried samples were homogenised, using a grinder, before extraction. The methanol extracts of E. cava were obtained by using previously described methods (Kang et al., 2010) with minor modifications. Briefly, dried E. cava powder (10 g) was dissolved in 70% MeOH (200 ml) and shaken for 24 h. After the extraction, the supernatant was recovered by centrifugation (7000 rpm) at 4 °C for 30 min and the methanol was evaporated using a rotary vacuum evaporator (Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The extract powder was suspended in distilled water and then partitioned with n-hexane, methylene chloride, ethyl acetate (EtOAc) in sequence. Each of these fractions was evaporated and kept on 20 °C until used. The total polyphenol contents and DPPH radical-scavenging activities of the fractions were determined according to methods previously described (Kang et al., 2010). 2.3. Measurement of total polyphenol content Total polyphenol content was determined by using the method Zhang et al. (2006) with a minor modification (Zhang et al., 2006). For polyphenol standard, a stock solution (1 mg/ml) of gallic acid (in distilled water) was prepared and the stock was diluted to give working standards of 0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 lg/ ml concentrations, respectively. For samples, an aliquot (5 mg) of several seaweeds methanol extract was dissolved in 1 ml of distilled water. To measure total polyphenol content, an aliquot (10 ll) of each sample or standard solution was mixed with 50 ll of Folin–Ciocalteu’s reagent in a 96-well microplate format and incubated for 5 min. The reactant was mixed with 40 ll of 7.5% sodium carbonate solution and incubated in a dark place for 2 h. Absorbance was measured at 750 nm with a spectrophotometric microplate reader (BioTek Instruments Inc., Winooski, USA). 2.4. DPPH radical-scavenging assay The antioxidant activity of E. cava methanol extract was determined using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method with a minor modification (Kang et al., 2010). For this, each fraction of E. cava extract was prepared as described above. A working

solution (6  105 M) of DPPH was also freshly prepared by dissolving it in DMSO and was vortex-mixed on the day of experiment. In brief, 900 ll of DPPH solution was mixed with 100 ll of distilled water (control), or 100 ll of each fraction of E. cava extract (sample). For the calibration of sample background, 100 ll of each fraction of E. cava extract was mixed with 900 ll of DMSO alone (sample background) instead of DPPH solution. Butylhydroxytoluene (BHT) and L-ascorbic acid were used as positive controls. An aliquot of control, sample solution, or sample background was transferred to a 96-well microplate and incubated for 1 h in a dark place. The absorbance at 517 nm was measured using a spectrophotometric microplate reader (BioTek Instruments Inc., Winooski, USA) and DPPH radical-scavenging activity of a sample was analysed using the equation as below.

DPPH radical-scavenging activity ð%Þ ¼ ½1ðA1  ðA2  A3 ÞÞ=A1   100

where, A1 is the absorbance of control; A2 is the absorbance of sample; A3 is the absorbance of sample background. 2.5. Cell culture A549 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 supplemented with 10% FBS, 100 lg/ ml of penicillin–streptomycin-amphotericin B solution at 37 °C in a 5% CO2-humidified incubator. Cells were passaged three times a week by treating with trypsin–EDTA and used for experiments after five passages. 2.6. MTT assay for cell viability Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) reduction assay, as described previously (Kang & Kim, 2010). Briefly, A549 cells were incubated at a density of 4  104 cells/well in 24-well plates for 24 h in 500 ll of RPMI 1640 with 10% FBS. The cells were treated with ECE (0–200 lg/ml) for 24, or 48 h. Then, MTT dye (5 mg/ml) was added to the cells and they were incubated for an additional 3 h. After the medium was removed, DMSO was added to the cells for the solubilisation of generated formazan salts. The amount of formazan salt was determined by measuring the optical density (OD) at 540 nm, using a GENiosÒ microplate spectrophotometer (PowerWaveTMXS, 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]  100). 2.7. Wound migration assay Cell migration assay was performed using 6-well plates, as previously described (Lee et al., 2006). A549 cells were seeded into 6-well plates (1  105 cells/ml) and grown to 80–90% confluence for the experiment. After aspirating the medium, cells were scraped with a sterile micropipette tip to create wound. They were washed twice with PBS to remove cellular debris and then replaced with complete RPMI 1640. A549 cells were treated with ECE (0, 12.5, 25, and 50 lg/ml) and incubated for 48 h. Cell migration into the wound area was photographed at the stages of 0 and 48 h, 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.

H. Lee et al. / Food Chemistry 127 (2011) 1229–1236

ing cells were removed from the upper surface of the filter membrane. The invading cells on the lower surface of the membrane were stained with crystal violet for 1 h and rinsed with water and dried. The amount of invading cells on the lower surface of the filter membrane was determined using a light microscope and NIH Image software (Image J).

2.8. Cell invasion assay The invasive behaviour of A549 cells was tested using a cell invasion chamber kit (BD Bioscience, San Jose, CA, USA) (Srikantan, Valladares, Rhim, Moul, & Srivastava, 2002). A549 cells were resuspended in a serum-free RPMI 1640 medium (5  104 cells/200 ll) in the absence or the presence of 0, 12.5, 25, and 50 lg/ml of ECE. The cells were seeded onto the upper chamber of a Matrigel-coated filter and a serum-containing 10% FBS (500 ll) was added to the lower chamber. After 48 h incubation, the non-invad-

2.9. Gelatin zymography MMP-2 secretion of A549 cells into culture medium was determined using 6-well plate gelatin zymography (Garbisa et al., 2001). Briefly, A549 cells were seeded (1  105 cells/well) and allowed to grow to confluence for 24 h and maintained in RPMI 1640 with 10% FBS. The cells were washed with PBS and incubated in ECE (0, 12.5, 25, and 50 lg/ml) in serum-free RPMI 1640 for 48 h. The supernatant was collected and mixed with non-reducing sample buffer, then electrophoresed in 10% polyacrylamide gel containing 0.1% (w/v) gelatin. After the electrophoresis, gel was washed for 30 min, twice, with 2.5% Triton X-100 and incubated for an additional 18 h at 37 °C for the enzymatic reaction of MMPs in zymography reaction buffer (200 mM NaCl, 10 mM CaCl2, 50 mM Tris–HCl, pH 7.4). The gel was then stained with Coomassie blue R-250 (0.125% Coomassie blue R-250, 50% methanol, 10% acetic

Table 1 Total polyphenol content and DPPH radical-scavenging activity of solvent fractions of E. cava. Fractions

Yield (%)

n-Hexane Methylene chloride Ethyl acetate Distilled water

2.54 1.65 20.2 43.7

A

Total polyphenol (lg/mg)

DPPH-scavenging activity at 5 mg/ml (%)

29.2 78.0

42.7 59.4

329 71.0

86.3 59.6

1231

1

3

2

4

Cell M Ce Mig gra atio on n (% %)

B *

* *

ECE (µg/ml) ( g/ml) Fig. 1. Effect of ECE on A549 cell migration in wound healing assay. (A) A549 cells were seeded into 6-well plates and grown to 90% confluence in RPMI containing 10% FBS. Cells were scratched with a sterile pipette tip and then treated with ECE 0 lg/ml (1), 12.5 lg/ml (2), 25 lg/ml (3), and 50 lg/ml (4). (B) The cell migration distance was determined by the level of wound closure using Image J analysis. The data shown are the means ± SD of six experiments. ⁄Significant difference from control group, p < 0.05.

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acid) and destained (methanol/acetic acid/water, 40/10/50, v/v/v). Gelatin-digested (cleared) bands, representing the activities of MMP-2, were quantified by densitometric analysis, using a Hewlett–Packard scanner and NIH Image software (Image J).

for 30 min) and the resulting supernatant was collected and determined for its protein concentration using a Bio-Rad protein assay reagent (Bio-Rad, CA, USA). The whole-cell lysates were mixed with electrophoresis sample buffer, and cooked at 95 °C for 5 min.

2.10. Preparation of cell lysates

2.11. Western blotting

After ECE treatments, A549 cells were rinsed twice with icecold PBS, and then treated with 100 ll 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 Na3VO4, 1 lg/ml of aprotinin, 1 lg/ml of leupeptin, 1 lg/ ml of pepstatin). The lysis reaction was performed on ice with rocking for 3 min and the cells were scraped, using a rubber policeman, into Eppendorf microcentrifuge tubes. The scraped cells were allowed to lyse for an additional 30 min on ice, with periodic vortexing. Cell debris was removed by centrifugation (22,000g, at 4 °C

Equal protein content (60 lg) samples of cell lysates were separated on 12% SDS–polyacrylamide gel, transferred to polyvinylidene difluoride membranes (Bio-Rad, CA, USA), and subsequently subjected to immunoblot analysis, using specific primary antibodies, overnight at 4 °C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA) for 1 h at room temperature. The blots were then visualised by using the enhanced chemiluminescence method (ECL, Amersham Biosciences, Buckinghamshire, UK) on blue light-sensitive film (Fujifilm Corporation,

A

1

2

3

4

C ll Inv Ce I vas sio on (% %)

B

* * *

ECE (µg/ml) Fig. 2. Anti-invasive effect of ECE on A549 cells. (A) Effects of ECE on cancer cell invasion were examined using a Cell Invasion Assay kit (BD Bioscience, San Jose, CA, USA). For this, A549 cells were seeded onto the upper chamber of Matrigel-coated filter and treated with ECE 0 lg/ml (1), 12.5 lg/ml (2), 25 lg/ml (3), and 50 lg/ml (4) for 48 h. The invading cells on the lower surface of the membrane were visualised by crystal-violet staining and observed with a light microscope. (B) The level of cell invasiveness was quantified using Image J software. The data shown are the means ± SD of three experiments. ⁄Significant difference from control group, p < 0.05.

H. Lee et al. / Food Chemistry 127 (2011) 1229–1236

Tokyo, Japan). If necessary, the blotted membranes were stripped and reprobed using other antibodies. Densitometry analysis was performed with a Hewlett–Packard scanner and NIH Image software (Image J). 2.12. Statistical analysis The results were expressed as means ± standard deviation (S.D.). Statistical analyses of data were done by one-way ANOVA, and P < 0.05 was considered to be statistically significant. 3. Results 3.1. ECE is a polyphenol-enriched fraction of E. cava and has strong high anti-oxidative activity Previous reports have suggested that E. cava has strong antioxidant activity and considerable amounts of polyphenol. The 70% methanol-extracted powder was suspended in distilled water and further partitioned with n-hexane, methylene chloride, ethyl acetate (EtOAc), in sequence. Then the total polyphenol contents and DPPH radical-scavenging activity of the fractions were determined (Table 1). Due to its strong anti-oxidative effect and high polyphenol contents, ECE was chosen and used throughout this study. First, the effect of ECE on A549 cell viability was examined at various concentrations. The treatment of ECE (0–100 lg/ml, for 24, 48 h) did not show any significant cytotoxic effect in the present experiments (data not shown). 3.2. ECE inhibits the migration and invasion of A549 cells Metastatic cancers have several important characteristics, including the migratory and invasive activities of tumour cells. To examine whether ECE has any inhibitory effect on cancer cell migration, A549 cells were incubated in the absence or presence of ECE for wound migration assay. As shown in Fig. 1A, ECE treatment strongly suppressed A549 cell migration to the wounded area

A 72kDa

MMP-2

0

12.5

25

50

/ ECE µ g/ml) C (µ

MMP-2 ac MM ctivitty (% %)

B * * *

1233

in a concentration-dependent manner. Comparing this result with that of vehicle-treated control, the incubation with ECE at 12.5, 25, and 50 lg/ml (for 48 h) significantly reduced the cancer cell migration by 24, 38, and 65%, respectively (Fig. 1B). For undergoing metastasis, cancer cells must degrade and cross extracellular matrix. Hence, the effect of ECE on cancer cell invasion was evaluated using invasion chamber assay, which was performed on A549 cells in the absence or the presence of various concentrations of ECE. From this, the penetration of A549 cells through the Matrigelcoated filter was significantly down-regulated by ECE in a concentration-dependent manner (Fig. 2A and B). These results suggest that ECE has strong inhibitory effects on the migration and invasion of A549 cells. 3.3. ECE inhibits the activation of MMP-2 in A549 cells High levels of MMPs secretion have been reported in various types of metastatic tumours. This process is believed to be associated with the degradation of ECM, which is essential for tumour metastasis. To determine whether ECE can inhibit the MMP-2 activity of A549 cells, the cancer cells were treated with various concentrations of ECE in serum-free medium for the indicated periods of time. Then the conditioned media of the cells were collected and their MMP-2 activity was assessed using the gelatin zymography method. As shown in Fig. 3A and B, ECE exhibited a concentration-dependent inhibitory effect on the MMP-2 activity of A549 cells (12, 31, and 69% of control at 12.5, 25, and 50 lg/ml of ECE, respectively). On the other hand, the impact of ECE on the MMP9 activity was inconclusive, since an extremely low level of MMP-9 was detected in A549 cells, even in the absence of ECE (data not shown). 3.4. ECE inhibits the phosphorylations of Akt and p38 Since ECE inhibits the migration and invasion of A549 cells, via inhibiting MMP-2 activity, the underlying mechanism was further investigated for its cell signalling pathways. Several reports suggest that MAPKs (JNK 1/2, ERK 1/2, p38) and PI3K–Akt are involved in cancer cell migration, invasion, and MMP-2 activity (Cheng, Chou, Kuo, & Hsieh, 2006). Hence, the effects of ECE on these signalling pathways were examined on A549 cells. For this, the cancer cells were treated for 3 h with various concentrations of ECE (0, 12.5, 25, and 50 lg/ml) (Fig 4A and B). Alternatively, A549 cells were treated with 50 lg/ml of ECE for the indicated periods of time (0, 1, 3, and 6 h) under similar conditions. The results showed that ECE treatment did not alter the phosphorylation levels of JNK 1/2 and ERK 1/2, regardless of concentration and time of treatment. In contrast, ECE noticeably decreased the phosphorylations of Akt and p38 in A549 cells. Comparing this result with vehicle-treated control (100%), the status of phospho-Akt was significantly decreased to 52 and 32% of control at 25 and 50 lg/ml of ECE, respectively (Fig. 4C and D). The level of phospho-p38 was also downregulated from 3 to 6 h of ECE treatment at high concentration (50 lg/ml) (Fig. 4E and F). 3.5. Inhibition of PI3K/Akt is a mechanism of action of ECE against A549 metastatic activity

ECE ((µg/ml) µg/ml) Fig. 3. Effect of ECE on MMP-2 activity of A549 cells. (A) Subconfluent A549 cells were incubated for 48 h in the absence or presence of various concentrations (12.5, 25, 50 lg/ml) of ECE in serum-free RPMI media. The conditioned media were collected, and their MMP-2 activities were determined using gelatin zymography. (B) MMP-2 activity was quantified by measuring the band intensities using Image J software. The data shown are the means ± SD of six experiments. ⁄Significant difference from control group, p < 0.05.

Apparently the ECE treatment of A549 cells resulted in a strong inhibition of Akt, suggesting that the PI3K/Akt pathway may be a critical target of ECE in prohibiting the metastatic activities. To confirm this hypothesis, A549 cells were co-treated with ECE and LY294002 (a PI3K inhibitor); then their MMP-2 activities were investigated (Fig. 5). Interestingly, the MMP-2 activity of A549 was synergistically suppressed by the co-treatment, in which the inhibitory effect (83%) was far greater than the summation of each

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H. Lee et al. / Food Chemistry 127 (2011) 1229–1236

A

B

µ g/ml) ECE (µ 0

12.5 12 5

25

50

50 µg/ml ECE (h) µg ( ) 0

1

3

6

54kDa

p-JNK1 p JNK1

46kDa

p-JNK2

p p-JNK1 p-JNK2 p JNK2

44kDa 42kDa

p-ERK1 p-ERK2 ERK2

p-ERK1 p-ERK2

43kDa

p-p38 p p

p-p38 p p

60kDa

p-Akt

p-Akt Ak

37kDa

GAPDH

GAPDH

* *

p--Ak kt (% ( off co ontro ol)

D

p--Ak kt (% (% off co on ntro ol)

C

ECE(µg/ml) (µg )

*

*

Time ((h))

*

* *

ECE( C (µg/ g/ml))

p--p3 38 (% % of con c ntro ol))

F

E p-p3 p 38 (% % of o c con ntrrol))

*

* *

Time ((h))

Fig. 4. Concentration- and time-dependent effects of ECE on JNK 1/2, ERK 1/2, p38, and Akt in A549 cells. (A, C, E) A549 cells were incubated for 3 h with ECE at various concentrations (0, 12.5, 25, and 50 lg/ml). The levels of phospho-JNK 1/2, phospho-ERK 1/2, phospho-p38, and phospho-Akt were analysed by western blotting using specific monoclonal antibodies. (B, D, F) A549 cells were treated with 50 lg/ml of ECE for the periods of 0, 1, 3, and 6 h, respectively. Western blotting was performed, as described above, and in Section 2. GAPDH was used as a loading control. (C, D, E, F) The band intensities (phosphorylation levels) of all signalling molecules were quantified by the analysis with Image J software. The data shown are the means ± SD of three independent experiments. ⁄Significant difference from control group, p < 0.05.

(17% by ECE alone, and 42% by LY294002 alone). From this, the PI3K/Akt pathway appears to be an important therapeutic target of ECE against the metastatic lung cancer cells. These results imply that ECE strongly modulates A549 cells invasion and MMP-2 activity by the mechanism involving PI3K/Akt and p38 signalling pathways. 4. Discussion Cancer metastasis is a complex process and highly associated with the increased activities of MMPs, which are essential for degrading neighbouring connective tissues for cancer cell invasion and migration. Interestingly, several studies have shown that reactive oxygen species (ROS) can enhance MMP induction and that treatment with antioxidants can block the event (Sikka Suresh,

2003; Storz, 2005). ROS have also been implicated in a variety of pathologies, including carcinogenesis, cardiovascular diseases, neurodegenerative diseases, and chronic inflammation (Closa & Folch-Puy, 2004; Koutsilieri et al., 2002). In cancer metastasis, ROS have been reported to modulate the activities of crucial signalling molecules, coupled with MMP expression and its activation (Lin, Liang, & Lin-Shiau, 1999). In fact, N-acetylcysteine (NAC), a ROS-scavenger, blocks tumour invasion and angiogenesis by inhibiting MMP production and pro-MMP activation (Goldman, 2000; Morini et al., 1999). There are also some natural antioxidants, such as epigallocatechin gallate (EGCG), lycopene and other tea polyphenols, which have been reported to inhibit migration and invasion, as well as MMP-2 activity, of tumour cells (Lin et al., 1999). Marine seaweeds and their extracts have proved good natural sources for discovering novel antioxidant compounds. Among

H. Lee et al. / Food Chemistry 127 (2011) 1229–1236

A

MMP-2

72kDa

LY294002 ( µM) ECE (µg/ml) ( / l)

10

20

12 5 12.

10

20

12.5 12 5

12 12.5 5

MMP--2 (% MM %)

B * *

* *

LY294002 ( µM) ECE (µg/ml)

10 12 5 12.

20

*

10

20

12 5 12.5

12 12.5 5

Fig. 5. Effects of ECE and LY294002 on MMP-2 activity of A549 cells. (A) A549 cells were seeded into 6-well plates and pretreated with LY294002 (0, 10, or 20 lM) for 30 min, then incubated for an additional 48 h in the absence or the presence of ECE (12.5 lg/ml). The media were collected, and MMP-2 activity was assessed by gelatin zymography. (B) MMP-2 activity was quantified by measuring the band intensities using Image J software. The data shown are the means ± SD of six experiments. ⁄Significant difference from control group, p < 0.05.

others, E. cava has been reported to contain abundant polyphenols, especially phloroglucinol derivatives (phloroglucinol, eckol, fucodiphloroethol G, phlorofucofuroeckol A, dieckol, 7-phloroeckol, and 6,6’-bieckol) (Li et al., 2004). In recent years, increasing attention has been paid to E. cava, due to its powerful antioxidant activity and potential therapeutic benefits for various disease conditions (Ahn et al., 2004; Kang et al., 2010; Kim et al., 2008). The anti-metastatic effect of E. cava extract and its mechanism of action on cancer cells, however, have not yet been properly investigated. Therefore, it was our primary goal, in the present study, to determine a major fraction of E. cava of dominant anti-metastatic effect and elucidate the related cell signalling pathways. For this, various solvent extracts (n-hexane, methylene chloride, ethyl acetate and water) of E. cava were examined for their polyphenol contents and antioxidant activities. According to Mahinda et al. (2006), who fractionated a 70% methanol extract of E. cava, using ethyl acetate, chloroform, and n-hexane, the ethyl acetate fraction exhibited the most significant antioxidative activity, as well as the highest total phenolic content. There is another report that, among various fractions (n-hexane, chloroform, ethyl acetate and nBuOH), the most important polyphenol derivatives of E. cava were isolated from the ethyl acetate fraction, which showed various biological activities, as well as the strongest antioxidant activity (Yong et al., 2009). Based on these and our results, the polyphenol-enriched ECE was chosen for further investigation in the present work. Cancer cell migration and invasion are two important steps in cancer metastasis. From our results, ECE treatment concentration-dependently suppressed the migration and invasion of A549 lung adenocarcinoma cells (nearly up to 60% compared with those of vehicle-treated controls) at the concentration of 50 lg/ml (Figs. 1 and 2). MMP-2 is overproduced by malignant cancer cells and plays a key role in cancer cell metastasis because cancer cells need degradation of extracellular matrix to spread to another site (Li et al., 2004; Mackay, Corbitt, Hartzler, & Thorgeirsson, 1990). In addition, mice lacking MMP-2 have a reduced tumour burden and decreased metastasis, as well as reduced tumour angiogenesis

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without developmental abnormalities (Ohno-Matsui, Uetama, Yoshida, & Hayano, 2003). From gelatin zymography assay, MMP-2 activity was also noticeably decreased upon after treatment with ECE in A549 cells (Fig. 3). The major mechanisms associated with MMP-2 regulation are PI3K/Akt and mitogen-activated protein kinase family member (MAPKs) pathways. The MAPKs play important regulatory roles in cell growth, differentiation, apoptosis and metastasis (ChanHui, 1998). MAPKs include ERK1/2, c-Jun N-terminal kinase/ stress-activated protein kinase (JNK/SAPK) and p38 MAPK. Activation of ERK1/2 and p38 is implicated in mechanisms for promoting the expression of MMPs that are important for cell proliferation, angiogenesis, and invasion (Westermarck & Kahari, 1999). Thus, inhibition of the MAPK signals may prevent angiogenesis, proliferation, invasion and metastasis for a wide range of tumours (Shin et al., 2000). Furthermore, the PI3K and Akt pathways play a critical role in MMPs regulation (Shukla et al., 2007). Activation of PI3K tends to activate the downstream main target Akt and the constitutive PI3K–Akt activation contributes to the progress of cancer to a highly invasive and potentially metastatic state (Carpenter & Cantley, 1996; Hollborn et al., 2007). In a recent study, PI3K/Akt is a major pathway involving cell invasion and metastasis of NSCLC (Kikuchi et al., 2008). Thus, PI3K–Akt and its associated regulatory signalling pathways are potential targets for therapeutic strategy and molecular-based approaches for management of cancer. In the present study, ECE treatment showed negligible effects on the phosphorylation of JNK 1/2 and ERK 1/2. However, it significantly suppressed the levels of phospho-Akt and phospho-p38 in time- and concentration-dependent manners (Fig. 4). Also the inhibition of Akt appears to be far greater than that of p38, implying that the PI3K/Akt pathway may be a critical target of ECE-mediated MMP inhibition. The involvement of the PI3K/Akt pathway was further supported by using a PI3K inhibitor (LY294002), which reduced MMP-2 secretion (Fig. 5). The co-treatment of ECE and LY294002 further suppressed the MMP-2 activity, suggesting that PI3K/Akt should be an important target of ECE for inhibiting the metastasis of A549 cells. Therefore, the combination treatment of ECE and LY294002 (a PI3K inhibitor) on A549 cells could synergistically down-regulate the MMP-2 activity. In conclusion, ECE, a polyphenol-rich extract of E. cava, showed a potent inhibitory effect on the metastatic activity of A549 cells, including the suppressions of migration and invasion, and the down-regulation of MMP-2 activity by the mechanism of inhibition of the PI3K/Akt signalling pathway. However, ECE needs further study in the near future for better understanding of mechanism of action, including the downstream transcription factors, metastasis regulators (TIMP, adhesion factors), and other signalling pathways (FAK, Ras, angiogenesis-related signals). References Ahn, G. N., Kim, K. N., Cha, S. H., Song, C. B., Lee, J., Heo, M. S., et al. (2007). Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage. European Food Research and Technology, 226(1), 71–79. Ahn, M. J., Yoon, K. D., Min, S. Y., Lee, J. S., Kim, J. H., Kim, T. G., et al. (2004). Inhibition of HIV-1 reverse transcriptase and protease by phlorotannins from the brown alga Ecklonia cava. Biological and pharmaceutical bulletin, 27(4), 544–547. Banerji, A., Chakrabarti, J., Mitra, A., & Chatterjee, A. (2004). Effect of curcumin on gelatinase A (MMP-2) activity in B16F10 melanoma cells. Cancer letters, 211(2), 235–242. Carpenter, C. L., & Cantley, L. C. (1996). Phosphoinositide kinases. Current Opinion in Cell Biology, 8(2), 153–158. Chan-Hui, P. (1998). Human mitogen-activated protein kinase mediates the stressinduced activation of mitogen-activated protein kinase cascades. The Biochemical Journal, 336, 599–609. Cheng, J. C. H., Chou, C., Kuo, M., & Hsieh, C. (2006). Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/ Akt/NF-jB signal transduction pathway. Oncogene, 25(53), 7009–7018.

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Antimetastatic activity of polyphenol-rich extract of Ecklonia cava through the inhibition of the Akt pathway in A549 human lung cancer cells.

An ethyl acetate extract (ECE) of a brown alga, Ecklonia cava, was examined for its anti-metastatic effect, using A549 human lung carcinoma cells. ECE...
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