Food Chemistry 126 (2011) 1708–1715

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Pinolenic acid inhibits human breast cancer MDA-MB-231 cell metastasis in vitro

Szu-Jung Chen a, Chih-Ping Hsu b, Chi-Wei Li c, Jui-Hua Lu a, Lu-Te Chuang c,⇑a Department of Radiation Oncology, Tao Yuan General Hospital, Tao-Yuan, Taiwanb Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwanc Department of Biotechnology, Yuanpei University, Hsinchu, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 May 2010Received in revised form 18 November 2010Accepted 10 December 2010Available online 14 December 2010

Keywords:Pinolenic acid (PNA)Prostaglandin E2 (PGE2)Type II cyclooxygenase (COX-2)MetastasisMDA-MB-231

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.12.064

⇑ Corresponding author. Address: Department of Bisity, 306 Yuanpei St., Hsinchu 300, Taiwan. Tel.: +8866102312.

E-mail address: ltchuang@mail.ypu.edu.tw (L.-T. C

Pinolenic acid (PNA), a naturally-occurring polyunsaturated fatty acid (PUFA), is found mainly in pineseeds. Although many studies have demonstrated beneficial effects of pine seed oil, there are no reportsof the biological effects of PNA on cancer metastasis. The objective of this study was to investigate theeffect of PNA on human breast cancer MDA-MB-231 cell proliferation and metastasis in vitro. We foundthat PNA did not affect cell viability and cell-matrix adhesion, but it inhibited cell metastasis by suppress-ing cell invasiveness and motility. Suppression could in part be associated with the modification of then�6 PUFA composition of cells by PNA which significantly decreased the percentage of arachidonic acid(AA) in phospholipids from 12.6% to 4.9%. The lower AA content of the cancer cells might result in lesssynthesis of prostaglandin E2 (PGE2), and subsequent down-regulation of inducible cyclooxygenase(COX-2) expression. Thus, PNA represents a potential anti-cancer agent.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Pinolenic acid (PNA; D5,9,12-18:3), an unusual D5-desaturatedmetabolite of linoleic acid (LA; D9,12-18:2), occurs mainly in seedsof conifer plants in the genus Pinus. For years, pine seeds or pine seedoil (PSO) have been traditionally consumed as a health food in cer-tain regions, such as China, Korea and Siberia. Based on results fromanimal studies, PSO significantly lowered blood pressure and exhib-ited hypocholesterolemic effects (Asset et al., 1999; Sugano, Ikeda,Wakamatsu, & Oka, 1994). No adverse effects on brain developmentwere observed when young rats were fed with PSO (Pasquier, Ratna-yake, & Wolff, 2001). Recently, a PNA-oriented commercial product,PinnoThin™, was reported to reduce food intake and appetite bycontrolling the release of cholecystokinin (CCK) and glucogen-likepeptide-1 (GLP-1) in a pilot double-blind placebo-controlled clinicaltrial (Hughes et al., 2008; Pasman et al., 2008). Dietary fats have beenstrongly associated with an increased incidence of breast cancer, andthere is evidence that some dietary essential fatty acids influencebreast carcinogenesis (de Deckere, 1999; Maillard et al., 2002; Rose,Connolly, & Coleman, 1996; Rose, Connolly, Rayburn, & Coleman,1995). For example, in a study carried out by Rose et al. (1995), eico-sapentaenoic acid (EPA; D5,8,11,14,17-20:5) and docosahexaenoicacid (DHA; D4,7,10,13,16,19-22:6) from fish oil decreased the riskof breast tumour development (Rose et al., 1995). In contrast, arachi-

ll rights reserved.

otechnology, Yuanpei Univer-3 5381183x8151; fax: +886 3

huang).

donic acid (AA; D5,8,11,14-20:4), an n�6 polyunsaturated fatty acid(PUFA), stimulated proliferation and metastasis of breast cancercells (Rose et al., 1995). PNA is an n�6 PUFA but it contains a poly-methylene group within the carbon skeleton, and this makes thePNA structure distinct from its positional isomer c-linolenic acid(GLA; D6,9,12-18:3), which has well-known anti-inflammatoryproperties (Matsuo et al., 1996; Sugano et al., 1994). Thus, wehypothesised that PNA might exert differential effects on mammarycarcinogenesis as compared to other PUFA.

Numerous investigations have shown that elevated concentra-tions of prostaglandin E2 (PGE2) and over-expression of the induc-ible cyclooxygenase (COX-2) are highly correlated with themalignant changes in mammary cell carcinogenesis, including pro-motion of cell proliferation and enhancement of cell motility andinvasiveness (Davies et al., 2003; Horia & Watkins, 2007; Larkins,Nowell, Singh, & Sanford, 2006). Proposed mechanisms wherebyEPA and DHA suppress human breast cancer cell proliferationand metastasis include reduction of the content of AA, increasedproduction of PGE2 and upregulation of COX-2 (Kundu, Yang,Dorsey, & Fulton, 2001; Rolland, Martin, Jacquemier, Rolland, &Toga, 1980). These hypotheses are supported by studies in whichboth cell culture and cancer cells were implanted in athymic nudemice models (Horia & Watkins, 2007; Rose et al., 1995, 1996).However, there is still a need to evaluate whether other PUFAmight also inhibit human cell carcinogenesis through such mecha-nisms. Previously, PNA and its elongated metabolites, such as D7-eicosatrienoic acid (D7-ETrA; D7,11,14-20:3) and D9-docosatrie-noic acid (D9-DTrA; D9,13,16-20:3), were shown to modulatethe fatty acid compositions of the phospholipids of macrophages

S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715 1709

and hepatoma cells (Chuang, Tsai, Lee, & Huang, 2009; Tanakaet al., 1999), mainly by reducing the percentage of arachidonicacid, the precursor of pro-inflammatory PGE2 in cellular phospha-tidylinositol (PI) (Tanaka et al., 1999). Furthermore, PNA (incorpo-rated into murine macrophages) competed with AA for COX-2 andresulted in a marked decrease in the production of PGE2 (Chuanget al., 2009). Since PNA significantly reduced cellular PGE2 produc-tion, this fatty acid could also modulate breast cancer cell prolifer-ation and metastasis. We were therefore interested in testing thehypothesis that PNA suppresses cancer metastasis.

To this end, we used an established culture of human breastcancer MDA-MB-231 cells to see whether PNA could modulate cellproliferation, adhesion, migration and motility. We further investi-gated whether PNA was incorporated into cellular phospholipids,and if such incorporation was associated with a decrease in theproportion of AA in phospholipids and the production of PGE2 inresponse to cells stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In addition, expression of COX-2 was also assessed,to evaluate the modulatory effect of PNA on cell metastasis. Thisstudy provides new insights regarding the role of PNA in modulat-ing mammary cell carcinogenesis.

2. Materials and methods

2.1. Chemicals

Trypan blue, 12-O-tetradecanoylphorbol 13-acetate (TPA), di-methyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT), Tween-20, triheptadecanoin,protease inhibitors, gelatin and BCIP/NBT substrate were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Gas chromatography (GC)standards RL-461 were obtained from Nu-Chek-Prep, Inc. (Elysian,MN). Authentic fatty acids, namely PNA, GLA, EPA and DHA andthe PGE2 assay kit were supplied by Cayman Chemicals (Ann Arbor,MI). Trizol reagent and SuperScript� III First-Strand SynthesisSuperMix were supplied by Invitrogen (Carlsbad, CA). Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM)and foetal bovine serum (FBS) were from Gibco (Carlsbad, CA). Allreagent-grade organic solvents were from Burdick & Jackson (Mus-kegon, MI).

2.2. Cell culture and growth conditions

The human breast cancer MDA-MB-231 cell line was obtainedfrom the Bioresource Collection and Research Centre (Hsinchu,Taiwan). Cells were cultured in DMEM supplemented with 10%(v/v) heat-inactivated FBS, and maintained in a 5% CO2 fullyhumidified environment at 37 �C. To eliminate interference causedby components in FBS, we used a serum-free medium, containing0.5% (w/v) fatty acid-free bovine serum albumin (Roche, Indianap-olis, IN) in most experiments. Cancer cells (1 � 104 cells/well) wereseeded in a 96-well culture plate, and allowed to adhere and growat 37 �C. After 24 h of incubation, the culture medium was replacedby serum-free DMEM medium supplemented with different con-centrations (50 or 100 lM) of DHA, EPA, GLA or PNA for a further24 h. To monitor cell viability following these treatments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the trypan blue dye-exclusion method were used, as describedelsewhere (Chuang et al., 2009).

2.3. Adhesion assay

Fatty acid pre-treated MDA-MB-231 cells (1 � 105 cells/well)were seeded in a 48-well BD BioCoat™ fibronectin plate (BD Biosci-

ences, San Jose, CA), and incubated for 1 h at 37 �C. The adherentcells were then rinsed twice with PBS and re-incubated with freshmedium containing MTT (0.5 mg/ml) for 2 h at 37 �C. TheMTT-containing medium was removed and replaced with 0.4 mlof 0.04 N HCl/isopropanol. After 10 min of incubation, the absor-bance of the samples was measured at 540 nm by using a micro-plate ELISA reader (EL-800, Bio-Tek, Winooski, VT).

2.4. Cell migration assay

MDA-MB-231 cells (1 � 106 cells/well) were seeded into a reus-able 8-well FlexiPERM small insert (Greiner Bio-One GmbH,Frickenhausen, Germany). After 18 h of incubation, cells wererinsed and incubated with fresh serum-free DMEM and 100 lMconcentrations of the test fatty acids for 24 h. The culture mediumwas then discarded and the cells were treated with TPA (10 ng/ml)for 2 h. Following TPA simulation, the small insert was removed.Cells were gently washed with PBS, twice, and re-incubated withfresh serum-free medium for 24 h, and digitally photographed.The area of migration was measured using the software ScionImage (Scion Corporation, Frederick, MD).

2.5. Invasion assay

The invasion assay was carried out, essentially as described byHsu and Chung (2006) in modified Boyden’s chambers constructedusing multi-well cell culture plates and inserts (Falcon, LincolnPark, NJ, USA) (Hsu & Chung, 2006). The upper chamber was coatedwith 0.01% (w/v) gelatin and air-dried for 16 h, which served as theinvasion barrier, whereas DMEM, with 10% (w/v) FBS, was placedin the lower chamber to provide the chemoattractant. Fatty acid-treated MDA-MB-231 cells (2 � 106 cells per chamber) were thenadded to the serum-free medium in the upper chamber and incu-bated at 37 �C for 24 h. The cells retained on the upper surface ofthe filter were removed by wiping with a filter paper. The filters,with invading cells on the lower surface, were stained with Diff-Quik stain solution (Dade-Behring; Deerfield, IL) and fixed onto aglass slide. Cells were counted in 10 randomly-selected micro-scopic fields (�400). Experiments were performed independently,three times.

2.6. Lipid extraction and fatty acid analysis

Fatty acid analysis was performed to determine fatty acid com-positions. Adherent cells (5 � 105 cells/ml) were incubated in theserum-free DMEM supplemented with 100 lM PUFA for 24 h. Afterincubation, cells were harvested by centrifugation, and the pelletswere rinsed twice with PBS. Total cellular lipids were then pre-pared according to the modified method described by Folch, Lees,and Sloane-Stanley (1957). Briefly, cell pellets were immersed with20 ml of chloroform/methanol (2:1, v/v) at room temperature for1 h. The extracted lipids in the chloroform phase were separatedfrom the aqueous phase by adding 4 ml of NaCl solution (0.9%,w/v). Total lipids were collected and evaporated to dryness at40 �C under a stream of nitrogen, and were re-constituted with0.25 ml of chloroform.

The phospholipids fraction of total cellular lipids was isolated bythin-layer chromatography (TLC), using a developing solvent mix-ture consisting of hexane/diethyl ether/acetic acid (70:30:1, v/v/v). Fatty acid methyl esters (FAME), derived from the phospholipidsfraction, were prepared and analysed by an Agilent 6890 gas chro-matograph equipped with a flame-ionisation detector (FID). Onemicrolitre aliquots of the sample were injected in splitless-modeonto a fused-silica capillary column (Omegawax; 30 m � 0.32 mm,i.d., film thickness 0.25 lm, Supelco, Bellefonte, PA). The tempera-ture of the injector was set at 205 �C, detector at 235 �C, oven at

1710 S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715

140 �C, initially, then increased to 205 �C at 6 �C per min, 205 �C for15 min. Helium was the carrier gas, and the flow rate was set at3.5 ml per min. A mixture of authentic FAME standards (RL-461,Nu-Chek-Prep, Inc., Elysian, MN) and the internal standard (trihep-tadecanoin, Sigma, St. Louis, MO) was used to identify and quantifythe fatty acids in the cellular phospholipids.

2.7. In vitro study of PGE2 production

MDA-MB-231 cells were seeded into 24-well plates at5 � 105 cells/well with DMEM medium containing 10% (w/v) FBSat 37 �C and allowed to adhere for 4 h. After 24 h of incubation withPNA or other PUFA, cells were stimulated with 10 ng/ml of TPA for24 h. The cell-free medium was collected and the concentration ofPGE2 was analysed using the PGE2 enzyme immunoassay kit, as de-scribed elsewhere (Horia & Watkins, 2007), where conditions foroptimising the PGE2 response to TPA were defined.

2.8. Detection of COX-2 expression by Western blot

Cancer cells (5 � 105/ml) were treated with 100 lM concentra-tions of each of the PUFA for 24 h, followed by 12 h of TPA stimu-lation (10 ng/ml). After harvesting cells, total cellular protein wasextracted, and its concentration was determined by the BCA Pro-tein Assay Reagent Kit (Pierce, Rockford, IL). Heat-denatured pro-tein samples were loaded and then separated on 10% (w/v)sodium dodecyl sulphate–polyacrylamide gel (SDS–PAGE) at150 V constant voltage for 1 h, and electrophoretically transferredonto polyvinylidene difluoride (PVDF) membranes (Millipore,Billerica, MA) at 400 mA constant current for 1 h at 4 �C. Afterblocking and rinsing, the membranes were probed with the pri-mary antibody (1:500 dilution of anti-mouse COX-2 antibody; BDBiosciences, Franklin Lakes, NJ), and then reacted with a secondaryantibody (1:5000 dilution of anti-mouse immunoglobulin-alkalinephosphatase; Sigma, St. Louis, MO). Following another washingwith PBS-T, the immuno-complexes of interest were visualisedby using BCIP/NBT.

2.9. Detection of COX-2 expression by reverse transcription-polymerase chain reaction

Total RNA of TPA-treated MDA-MB-231 cells was isolated usingthe TRIzol reagent, and cDNA was synthesized from 1 lg of totalRNA, using the SuperScript� III First-Strand Synthesis SuperMixKit. To amplify cDNA, primer sets for COX-2 (forward primer, 50-GAATGGGGTGATGAGCAGTT-30; reverse primer, 50-CAGAAGGG-CAGGATACAGC-30) and glyceraldehyde 3-phosphate dehydroge-nase (GAPDH) (forward primer, 50-CATGGAGAAGGCTGGGGCTC-30; reverse primer, 50-CACTGACACGTTGGCAGTGG-30) were appliedfor PCR. The expression of human GAPDH was examined as aninternal control. PCR amplification was run on a DNA thermal cycler(Applied Biosystem, Foster City, CA), following the programme of30 s at 94 �C, 30 s at 55 �C, and 30 s at 72 �C for 30 cycles, followedby extension for 5 min at 85 �C. The PCR products were separatedon 2% (w/v) agarose gels containing ethidium bromide. Bands cor-responding to respective specific PCR products were quantified bydensitometric scanning and analysed by the Gel-Pro Analyzer(Bethesda, MD).

2.10. Statistical analyses

Data were analysed by analysis of variance (ANOVA) andDuncan’s multiple range test, using SPSS software (SPSS for win-dows 10.0; SPSS Inc. Chicago, IL) to determine differences betweenmeans. Means’ differences were considered significant at theP 6 0.05 levels.

3. Results

3.1. Effect of PNA on proliferation and adhesion

In this study, we determined whether PNA could modulatebreast cancer cell proliferation and metastasis. To examine the de-gree to which cell proliferation and metastasis were affected byPNA, we also treated the cells with n�3 and n�6 PUFA that havewell-documented anti-carcinogenetic effects in vitro, includingDHA, EPA and GLA. When MDA-MB-231 cells were treated with50 or 100 lM PNA, GLA or EPA, viabilities of the tested cells werenot statistically different from the untreated control (Fig. 1A).However, 100 lM DHA significantly decreased cancerous cell pro-liferation. With regard to whether PNA or other PUFA could influ-ence cell-matrix adhesion, which is the first stage of cellmetastasis, no significant effect was observed when cancer cellswere treated with 100 lM concentrations of any of the fatty acids(Fig. 1B).

3.2. Effect of PNA on cell motility and invasion

The effects of PNA and the three other fatty acids on the motilityand invasiveness of MDA-MB-231 cells were examined. The resultsin Fig. 2 show that the area occupied by cells treated with PNA,DHA or EPA was less than that occupied by the TPA-stimulatedcontrol. GLA, however, did not inhibit cell migration. Since bothPNA and DHA inhibited cell motility, we further investigatedwhether these two fatty acids might also inhibit the penetratingability of the MDA-MB-231 cells in the matrix assay. Supplement-ing cells with DHA or PNA significantly decreased cell invasion by29.6% and 25.4%, respectively (Fig. 3).

3.3. Effect of PNA on cellular PUFA compositions

Incorporation of exogenous fatty acids into the culture mediuminfluenced cellular fatty acid composition. To examine the uptakeand metabolism of PNA and other PUFA, MDA-MB-231 cells wereincubated for 24 h in serum-free medium supplemented separatelywith 50 lM concentrations of each of the fatty acids. Results fromGC analysis show that a higher portion of PNA was taken up bythe cells, and incorporated and metabolised to form D7-ETrA andD9-DTrA (Table 1). The incorporation of PNA and its elongatedmetabolites into cellular phospholipids significantly decreasedthe percentages of LA, dihomo-c-linolenic acid (DGLA; D8,11,14-20:3), AA, ADA and n�6 dicosapentaenoic acid (n�6 DPA;D4,7,10,13,16-22:5) (Table 1), especially AA, from 12.6% to 4.9% rel-ative to the control. Similar results were observed when cells wereincubated with DHA or EPA. Greater incorporation of DHA (39.0%)or EPA and its metabolite, n�3 DPA (D7,10,13,16,19-22:5)(40.0%), into cellular phospholipids occurred, and decreased thepercentage of AA from 12.6% to 5.7%. In contrast, when MDA-MB-231 cells were incubated with GLA, the incorporated GLA wasmetabolised rapidly to DGLA (25.9%) and subsequently to AA(12.7%).

3.4. Effect of PNA on prostaglandin E2 production

The results in Table 1 demonstrate that incubation of breastcancer cells with DHA, EPA or PNA significantly diminished theproportions of AA in cellular phospholipids. Since AA is the precur-sor of PGE2, it is logical to speculate that the decrease in the pro-portion of AA in cellular phospholipids might lead to a reductionin PGE2 synthesis. To test this hypothesis, MDA-MB-231 cells wereincubated with the serum-free medium containing 50 and 100 lMconcentrations of each of the four fatty acids for 24 h, following

B

0

20

40

60

80

100

120

Ctrl DHA EPA GLA PNA

Cel

l ad

hes

ion

(%)

0

20

40

60

80

100

120

Ctrl DHA

50 µM 100 µM

EPA

50 µM 100 µM

GLA

50 µM 100 µM

PNA

50 µM 100 µM

Cel

l via

bili

ty (

%)

Aa,c a a

a,c a,ca,c c

b,c

b

Fig. 1. Effect of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), c-linolenic acid (GLA) or pinolenic acid (PNA) on MDA-MB-231 cell proliferation (A) and cell-matrixadhesion (B). To determine cell proliferation: MDA-MB-231 cells were planted at a density of 1 � 104 cells/well in a 96-well plate with DMEM supplemented with 10% FBS for24 h. After 24 h of incubation, the culture medium was replaced by serum-free DMEM medium supplemented with 0.5% fatty acid-free bovine serum albumin and twoconcentrations (50 or 100 lM) of respective fatty acids for 24 h. Cell viability was estimated by the MTT assay. To determine cell adhesion: PUFA-treated (100 lM) MDA-MB-231 cells (1 � 105 cells/well) were seeded in a 48-well fibronectin-coated plate and incubated for 1 h. The culture medium was discarded, and adherent cells were detected bythe MTT assay. Each bar is the mean ± SD of three independent experiments. The values with different letters are significantly different from each other at P < 0.05.

S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715 1711

treatment with TPA for 24 h to stimulate the PGE2 synthesis. Theresults shown in Fig. 4 confirm that PGE2 synthesis was signifi-cantly enhanced by TPA stimulation in the control cells. Cells trea-ted with 50 or 100 lM concentrations of PNA, DHA or EPA secretedless PGE2 than did the TPA-stimulated controls, and the reductionwas dose-dependent (Fig. 4). When cells were incubated with50 lM GLA, the PGE2 production was slightly decreased; however,PGE2 synthesis was increased by inclusion of 100 lM GLA in theculture medium. This experiment demonstrated that PNA wasthe most potent of the fatty acids in reducing PGE2 productionby the cancer cells.

3.5. Effect of PNA on gene expression of COX-2

To understand the mechanism underlying the inhibitory effectof PNA on the synthesis of PGE2, we examined how PNA or otherPUFA might affect gene expression of COX-2 by the cancer cells.The results in Fig. 5 show that COX-2 protein expression, at thetranscriptional and translational levels, was significantly decreasedwhen cells were incubated with 100 lM concentrations of PNA,DHA or EPA. Furthermore, there was no statistical difference inthe levels of COX-2 expression amongst these three fatty acid-trea-ted samples. In contrast, no such inhibitory effect of GLA on COX-2expression, at both the protein and mRNA levels, was observed.

4. Discussion

The most important finding of this study was that PNA incorpo-ration modulated PGE2 production and COX-2 expression, andmarkedly inhibited cell metastasis through the suppression of bothcell invasiveness and motility; however, this unusual oil seed-derived special fatty acid did not affect human breast MDA-MB-

231 cell proliferation in vitro. Long-chain PUFA have been reportedto exert differential effects on cancer cell proliferation (Hawkins,Sangster, & Arends, 1999; Schley, Jijon, Robinson, & Field, 2005).In the present study, incubation of cells with DHA significantlylowered MDA-MB-231 cell proliferation, and the inhibitory effectwas dose-dependent (Fig. 1A). With regard to possible mechanismsunderlying the decreasing viability of cancer cells, published re-ports suggest that peroxidation products of n�3 PUFA, especiallyDHA, may cause cytotoxicity or affect intracellular transductionsignalling, resulting in the inhibition of cell growth or proliferation(Ding, Vaught, Yamauchi, & Lind, 2004; Gonzalez et al., 1991).However, in the present study, 100 lM PNA, GLA or EPA had no ef-fect on cell proliferation (Fig. 1A). On the other hand, cell viabilitydeclined by more than 90% as the concentration of each of the fattyacids in the culture medium reached 300 lM (data not shown). Wefound, as noted by other investigators, that DHA was more anti-proliferative than were EPA or GLA (Barascu, Besson, Floch,Bougnoux, & Jourdan, 2006; Horia & Watkins, 2007; Schley et al.,2005). The observations in this study also indicate that PNA is aless potent modulator of cell proliferation than is DHA.

Tumour metastasis is a multiple-stage process that involves celladhesion, invasion, migration and angiogenesis. Initially, cancercells, which have detached from the primary tumour, interact withcomponents of the extracellular matrix (ECM) through cell-matrixadhesion molecules. Once metastatic cells adhere to the surround-ing basement membrane (BM), they secrete specialised proteasesthat degrade connective tissue and initiate the process of cell inva-sion and migration (Stetler-Steveson, Aznavoorian, & Liotta, 1993).Therefore, interrupting at least one of these processes represents arational approach to preventing tumour formation. In the presentstudy, although the fatty acid composition of the phospholipidsof MDA-MB-231 cells was modified by incubation with PUFA

0 h 24 h

Ctrl

DHA

EPA

GLA

PNA

0

20

40

60

80

100

120

Ctrl DHA EPA GLA PNA

Are

a o

f m

igra

tio

n (

% o

f C

trl)

aa

b

b b

A

B

Fig. 2. Effect of PNA, DHA, EPA or GLA on MDA-MB-231 cell migration. The cells were seeded in an 8-well small insert at a density of 1 � 106 cells/well and incubated withserum-containing medium for 18 h. After incubation, the medium was removed and replaced with the serum-free DMEM and 100 lM concentration of respective fatty acidfor 24 h, followed by 2 h of 12-O-tetradecanoylphorbol 13-acetate (TPA) (10 ng/ml) stimulation. After TPA stimulation, the small insert was removed. Cells were re-incubatedwith fresh serum-free medium for 24 h and photographed under an inverted microscope. Each bar represents the mean ± SD of three independent experiments. The valueswith different letters are significantly different from each other at P < 0.05.

Ctrl DHA PNA

0

20

40

60

80

100

120

Ctrl DHA PNA

Cel

l inv

asio

n (

% o

f C

trl)

* *

Fig. 3. Effect of PNA or DHA on MDA-MB-231 cell invasion. Invasiveness of PNA- and DHA-treated MDA-MB-231 was assessed by invasion assay, using a Boyden chamber, asdescribed in Section 2. Each bar represents the mean ± SD of three independent experiments. The values with a symbol are significantly different from each other at P < 0.05.

1712 S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715

(Table 1), none of the four PUFA we tested affected cell-matrixadhesion to fibronectin (Fig. 1B). A previous report demonstratedthat short-term (2 h) incubation of exogenous GLA, EPA or DHAwith human breast cancer cells (MCF-7 and MDA-MB-231) signif-

icantly decreased the adhesion of the cells to type IV collagen,fibronectin and matrigel (Menéndez et al., 2002). When the resultsof the two studies are compared, it seems that the inhibitory effectof PUFA on cell adhesion might not be the result of the incorpora-

Table 1Fatty acid compositions (wt.%) of total phospholipids from fatty acid-treated MDA-MB-231 cells. LA, linoleic acid; GLA, c-linolenic acid; DGLA, dihomo-c-linolenic acid; AA,arachidonic acid; ADA, adrenic acid; n�6 DPA, n�6 docosapentaenoic acid; EPA, eicosapentaenoic acid; n�3 DPA, n�3 docosapentaenoic acid; DHA, docosahexaenoic acid; NMIFA,non-methylene-interrupted fatty acids; PNA, pinolenic acid; D7-ETrA, D7-eicosatrienoic acid; D9-DTrA, D9-docosatrienoic acid. MDA-MB-231 cells were incubated with serum-free medium alone, or medium supplemented with 50 lM concentrations of respective fatty acids (DHA, EPA, GLA or PNA) for 24 h. Each value represents the mean ± SD of threeindependent experiments. The values with different letters are significantly different from each other at P < 0.05.

Fatty acid treatments

Ctrl DHA EPA GLA PNA

n�6 PUFA18:2 (LA) 2.97 ± 0.02a 2.02 ± 0.02b 2.23 ± 0.17b 1.94 ± 0.06b 1.83 ± 0.07b

18:3 (GLA) 0.09 ± 0.01a 0.05 ± 0.01a 0.06 ± 0.01a 3.97 ± 0.43b 0.06 ± 0.01a

20:3 (DGLA) 1.76 ± 0.02a 1.10 ± 0.02b 1.12 ± 0.04b 25.9 ± 0.92c 1.00 ± 0.07b

20:4 (AA) 12.6 ± 0.33a 5.71 ± 0.07b 5.73 ± 0.35b 12.7 ± 0.09a 4.91 ± 0.03c

22:4 (ADA) 4.01 ± 0.05a 1.88 ± 0.05b 2.11 ± 0.12b 6.60 ± 0.35c 3.49 ± 0.06d

22:5 (n�6 DPA) 2.09 ± 0.02a 0.85 ± 0.05b 0.86 ± 0.09b 1.63 ± 0.08c 1.14 ± 0.02d

n�3 PUFA20:5 (EPA) 0.26 ± 0.03a 1.35 ± 0.01b 10.9 ± 2.34c 0.11 ± 0.01d 0.10 ± 0.01d

22:5 (n�3 DPA) 1.44 ± 0.01a 2.70 ± 0.06b 28.1 ± 1.53c 0.82 ± 0.02d 0.84 ± 0.01d

22:6 (DHA) 2.63 ± 0.09a 39.0 ± 0.63b 2.01 ± 0.21a 1.37 ± 0.01c 1.38 ± 0.02c

NMIFA18:3 (PNA) n.d. n.d. n.d. n.d. 13.1 ± 0.9220:3 (D7-ETrA) n.d. n.d. n.d. n.d. 29.3 ± 0.3422:3 (D9-DTrA) n.d. n.d. n.d. n.d. 2.41 ± 0.12

0

200

400

600

800

1000

1200

Ctrl DHA

50 µM 100 µM

EPA

50 µM 100 µM

GLA

50 µM 100 µM

PNA

50 µM 100 µM

PG

E2 C

on

c. (

pg

/ml)

Neg Pos

a

b

c

d

c,e

d

e

f

a

g

Fig. 4. Effects of PNA, DHA, EPA or GLA on the production of prostaglandin E2 (PGE2) in MDA-MB-213 cells. Cells were incubated with only medium, or medium containing 50or 100 lM concentrations of PNA, DHA, EPA or GLA for 24 h, followed by TPA treatment for 24 h. Each value represents the mean ± SD of three independent experiments.Values with different letters are significantly different from each other at P < 0.05.

Fig. 5. Effects of PNA, DHA, EPA or GLA on the expression of cyclooxygenase-2 (COX-2) at translational level (A) and transcriptional level (B) in MDA-MB-213 cells. Cells wereincubated with only medium, or medium containing 100 lM concentrations of PNA, DHA, EPA or GLA for 24 h, followed by TPA treatment for 12 h. COX-2 expression attranslational level was determined by Western blot analysis; b-actin was used as a loading control. The levels of COX-2 mRNA were estimated by RT-PCR assay. Each valuerepresents the mean ± SD of three independent experiments. Values with different letters are significantly different from each other at P < 0.05.

S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715 1713

tion of fatty acids into cellular phospholipids. An alternative mech-anism could involve PUFA modulation of second messenger sys-tems involving cell signalling, possibly through the down-regulation of the expression of protein kinases A and C (Moore,Wang-Johanning, Chang, & Johanning, 2001).

In contrast to cell adhesion, cell motility was significantly re-duced when MDA-MB-231 cells were pre-treated with PNA orn�3 PUFA (Fig. 2). Furthermore, PNA and DHA both suppressedthe invasiveness of cancer cells by inhibiting cell penetrationthrough the membrane filter, as previously described (Horia & Wat-

1714 S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715

kins, 2007; Menéndez et al., 2002). These findings suggest that PNAsuppresses cell metastasis by reducing cell motility and invasive-ness, rather than by modifying the cell adhesion to the cell-matrix.

The other aim of this study was to determine whether PNAcould modify fatty acid composition of cellular phospholipids inMDA-MD-231 cells, and thereby modulate the production ofPGE2. Fatty acid analysis of cellular phospholipids confirmed thatexogenous PNA and n�3 PUFAs were taken up by the cancer cellsand substituted, to a considerable extent, for AA in cellular phos-pholipids, resulting in reduced PGE2 production as we reportedpreviously (Chuang et al., 2009). The suppression of COX-2expression at both the translational and transcriptional levels byPNA, DHA or EPA seems to account, in part at least, for the de-crease in PGE2 biosynthesis (Fig. 5). This finding confirms thefinding of Horia and Watkins (2007) that n�3 PUFA reducesCOX-2 expression (Horia & Watkins, 2007); however, it was notin accordance with earlier results obtained using the RAW264.7macrophage model of inflammation (Chuang et al., 2009). Thelack of agreement between the human epithelial cell and murinemacrophage studies could be due to species differences. Compar-ing PGE2 released from cells treated with PNA or n�3 PUFA, thePNA treatment decreased PGE2 synthesis more than did the twon�3 PUFA. This decrease could be due to the lower cellular con-tent of AA when cells were treated with PNA (Table 1), or to thepresence of D7-ETrA (an elongation product of PNA) that com-petes more effectively with AA for the enzyme COX-2, but whichitself cannot be metabolised (Chuang et al., 2009). In contrast,GLA, a positional isomer of PNA, did not reduce PGE2 biosynthesisor COX-2 expression. These results could be accounted for if alarge fraction of the GLA that was taken up by the cells had beeneliminated by metabolism to DGLA or some other n�6 PUFA, suchas AA. The newly formed AA compensated those which were re-placed by GLA incorporation, and resulted in no significantchange in the proportion of AA (Table 1) and subsequent PGE2

production (Fig. 4). Furthermore, the percentage of DGLA wastwofold higher than that of AA in the phospholipid fraction(27.5% vs. 13.5%), suggesting that PGE1 may have been synthe-sized preferentially. PGE1 is potent anti-inflammatory agent, buthas no modulatory effect on cell metastasis. Based on our results,GLA did not appear to inhibit cell invasiveness or motility (Figs. 2and 3).

In this study, we demonstrated that PNA, EPA and DHA signifi-cantly reduced PGE2 production and COX-2 expression, and thatthese three fatty acids suppress breast cancer cell invasivenessand migration in vitro. These findings are consistent with earlier re-ports that show that PGE2 and COX-2 are highly associated withcancer cell metastasis (Davies et al., 2003; Horia & Watkins,2007; Larkins et al., 2006), suggesting that PNA might inhibit met-astatic MDA-MB-231 cells by modulating these two risk factors.Furthermore, recent evidence strongly implicates matrix metallo-proteinases (MMP), MMP-2 and MMP-9 in particular, as mediatorsof invasion and metastasis in certain malignant cancers (Larkinset al., 2006). It has been demonstrated that n�3 PUFA inhibitMMP-9 production and subsequent metastasis in human breastcancer cell lines (Liu & Rose, 1995). In addition, n�6 PUFA stimu-late the production of MMP-9, and this stimulatory effect has beenattributed to eicosanoid synthesis from AA (Liu & Rose, 1994).Interestingly, although PNA is an n�6 PUFA, the unique polymeth-ylene-interrupted structure might hinder PNA or D7-ETrA frombeing metabolised into PGE2. Thus, the incorporation of thesetwo non-methylene interrupted fatty acids (NMIFA) into cellularphospholipids modified n�6 PUFA metabolism, resulting in areduction of the concentrations of AA and PGE2. The decreased le-vel of PGE2 down-regulated COX-2 expression, and subsequentlyled to the possible decrease in the production of MMP (Tsujii,Kawano, & Dubois, 1997).

In conclusion, we have demonstrated that PNA markedly sup-pressed MDA-MB-231 cell invasiveness and motility, but withoutaffecting cell proliferation or cell-matrix adhesion. These effectswere due, in part, to the substitution of AA by PNA in cellular phos-pholipids, resulting in reduced PGE2 synthesis and lower levels ofCOX-2 protein and mRNA expression. Thus, PNA may act as a po-tential anti-cancer agent in lowering the risk of breast cancer byeffectively antagonizing PGE2 and COX-2 expression.

Acknowledgements

We thank Dr. Robert H. Glew for helpful comments and Englishcorrection of the manuscript. This work was partially supported bya research Grant (PTH9926) from the Tao Yuan General Hospital,Department of Health, The Executive Yuan, Taiwan.

References

Asset, G., Staels, B., Wolff, R. L., Bauge, E., Madj, Z., Fruchart, J. C., et al. (1999). Effectsof Pinus pinaster and Pinus koraiebsis seed oil supplementation on lipoproteinmetabolism in the rat. Lipids, 34, 39–44.

Barascu, A., Besson, P., Floch, O. L., Bougnoux, P., & Jourdan, M.-L. (2006). CDK1-cyclin B1 mediates the inhibition of proliferation induced by omega�3 fattyacids in MDA-MB-231 breast cancer cells. International Journal of Biochemistryand Cell Biology, 38, 196–208.

Chuang, L.-T., Tsai, P.-J., Lee, C.-J., & Huang, Y.-S. (2009). Uptake and incorporation ofpinolenic acid reduces n�6 polyunsaturated fatty acid and downstreamprostaglandin formation in murine macrophage. Lipids, 41, 217–224.

Davies, G., Salter, J., Hills, M., Martin, L.-A., Sacks, N., & Dowsett, M. (2003).Correlation between cyclooxygenase-2 expression and angiogenesis in humanbreast cancer. Clinical Cancer Research, 9, 2651–2656.

de Deckere, E. A. M. (1999). Possible beneficial effect of fish and fish n�3polyunsaturated fatty acids in breast and colorectal cancer. European Journalof Cancer Prevention, 8, 213–221.

Ding, W.-Q., Vaught, J. L., Yamauchi, H., & Lind, S. E. (2004). Differential sensitivity ofcancer cells to docosahexaenoic acid-induced cytotoxicity: The potentialimportance of down-regulation of superoxide dismutase 1 expression.Molecular Cancer Therapeutics, 3, 1109–1117.

Folch, J., Lees, M., & Sloane-Stanley, G. H. (1957). A simple method for the isolationand purification of total lipids from animal tissues. Journal of BiologicalChemistry, 226, 497–509.

Gonzalez, M. J., Schemmel, R. A., Gray, J. I., Dugan, L., Jr., Sheffield, L. G., & Welsch, C.W. (1991). Effect of dietary fat on growth of MCF-7 and MDA-MB231 humanbreast carcinomas in athymic nude mice: Relationship between carcinomagrowth and lipid peroxidation product levels. Carcinogenesis, 12, 1231–1235.

Hawkins, R. A., Sangster, K., & Arends, M. J. (1999). The apoptosis-inducing effects ofpolyunsaturated fatty acids (PUFAs) on benign and malignant breast cellsin vitro. Breast, 8, 16–20.

Horia, E., & Watkins, B. A. (2007). Complementary actions of docosahexaenoic acidand genistein on COX-2, PGE2 and invasiveness in MDA-MB-231 breast cancercells. Carcinogenesis, 28, 809–815.

Hsu, C.-P., & Chung, Y.-C. (2006). Influence of interleukin-6 on the invasiveness ofhuman colorectal carcinoma. Anticancer Research, 26, 4607–4614.

Hughes, G. M., Boyland, E. J., Williams, N. J., Mennen, L., Scott, C., Kirkham, T. C., et al.(2008). The effect of Korean pine nut oil (PinnoThin™) on food intake, feedingbehaviour and appetite: A double-blind placebo-controlled trial. Lipids in Healthand Disease, 7, 6.

Kundu, N., Yang, Q., Dorsey, R., & Fulton, A. M. (2001). Increased cyclooxygenase-2(COX-2) expression and activity in a murine model of metastatic breast cancer.International Journal of Cancer, 94, 681–686.

Larkins, T. L., Nowell, M., Singh, S., & Sanford, G. L. (2006). Inhibition ofcyclooxygenase-2 decreases breast cancer cell motility, invasion and matrixmetalloproteinase expression. BMC Cancer, 6, 181.

Liu, X.-H., & Rose, D. P. (1994). Stimulation of type IV collagenase expression bylinoleic acid in a metastatic human breast cancer cell line. Cancer Letters, 76,71–77.

Liu, X.-H., & Rose, D. P. (1995). Suppression of type IV collagenase in MDA-MB-435human breast cancer cells by eicosapentaenoic acid in vitro and in vivo. CancerLetters, 92, 21–26.

Maillard, V., Bougnoux, P., Ferrari, P., Jourdan, M. L., Pinault, M., Lavillonniere, F.,et al. (2002). N�3 and n�6 fatty acids in breast adipose tissue and relative riskof breast cancer in a case-control study in Tours, France. International Journal ofCancer, 98, 78–83.

Matsuo, N., Osada, K., Kodama, T., Lim, O., Nakao, A., Yamada, K., et al. (1996). Effectsof c-linolenic acid and its positional isomer pinolenic acid on immuneparameters of Brown-Norway rats. Prostaglandins Leukotrienes and EssentialFatty Acids, 55, 223–229.

Menéndez, J. A., del Mar Barbacid, M., Montero, S., Ropero, S., Escrich, E., Cortés-Funes, H., et al. (2002). Effects of dietary fatty acids on the proliferation,adhesion and metastatic potential of breast cancer cells: An experimentalreview. Clinical and Translational Oncology, 4, 77–84.

S.-J. Chen et al. / Food Chemistry 126 (2011) 1708–1715 1715

Moore, N. G., Wang-Johanning, F., Chang, P. L., & Johanning, G. L. (2001). Omega � 3fatty acids decrease protein kinase expression in human breast cancer cells.Breast Cancer Research and Treatment, 67, 279–283.

Pasman, W. J., Heimerikx, J., Rubingh, C. M., van den Berg, R., O’Shea, M., Gambelli, L.,et al. (2008). The effect of Korean pine nut oil on in vitro CCK release, onappetite sensations and on gut hormones in post-menopausal overweightwomen. Lipids in Health and Disease, 7, 10.

Pasquier, E., Ratnayake, W. M. N., & Wolff, R. L. (2001). Effects of D5polyunsaturated fatty acids of maritime pine (Pinus pinaster) seed oil on thefatty acid profile of the developing brain of rats. Lipids, 36, 567–574.

Rolland, P. H., Martin, P. M., Jacquemier, J., Rolland, A. M., & Toga, M. (1980).Prostaglandin in human breast cancer: Evidence suggesting that an elevatedprostaglandin production is a marker of high metastatic potential for neoplasticcells. Journal of National Cancer Institute, 64, 1061–1070.

Rose, D. P., Connolly, J. M., Rayburn, J., & Coleman, M. (1995). Influence of dietscontaining eicosapentaenoic or docosahexaenoic acid in growth and metastasisof breast cancer cells in nude mice. Journal of National Cancer Institute, 87,587–592.

Rose, D. P., Connolly, J. M., & Coleman, M. (1996). Effect of omega�3 fatty acids onthe progression of metastases after the surgical excision of human breast cancercell solid tumors growing in nude mice. Clinical Cancer Research, 2, 1751–1756.

Schley, P. D., Jijon, H. B., Robinson, L. E., & Field, C. J. (2005). Mechanisms ofomega�3 fatty acid-induced growth inhibition in MDA-MB-231 human breastcancer cells. Breast Cancer Research and Treatment, 92, 187–195.

Stetler-Steveson, W. G., Aznavoorian, S., & Liotta, L. (1993). Tumor cell interactionswith the extracellular matrix during invasion and metastasis. Annual Review ofCell Biology, 74, 541–573.

Sugano, M., Ikeda, I., Wakamatsu, K., & Oka, Y. (1994). Influence of Korean pine(Pinus koraiensis)-seed oil containing cis-5, cis-9, cis-12-octadecatrienoic acid onpolyunsaturated fatty acid metabolism, eicosanoid production and bloodpressure. British Journal of Nutrition, 72, 775–783.

Tanaka, T., Takimoto, T., Morishige, J., Kikuta, Y., Sugiura, T., & Satouchi, K. (1999).Non-methylene-interrupted polyunsaturated fatty acids: Effective substitutefor arachidonate of phosphatidylinositol. Biochemical and Biophysical ResearchCommunications, 264, 683–688.

Tsujii, M., Kawano, S., & Dubois, R. N. (1997). Cyclooxygenase-2 expression inhuman colon cancer cells increases metastatic potential. Proceedings of theNational Academy of Sciences USA, 94, 3336–3340.