β-Catenin pathway and suppressed breast cancer cells in vitro and in vivo

Available online at www.sciencedirect.com

ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 104 – 110

RESEARCH ARTICLES

Docosahexaenoic acid inhibited the Wnt/β-Catenin pathway and suppressed breast cancer cells in vitro and in vivo☆ Meilan Xue a,⁎, Qing Wang b , Jinglan Zhao c , Liyan Dong d, Yinlin Ge a , Lin Hou a , Yongchao Liu a , Zheng Zheng a a

Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao, Shandong Province, 266021, China b Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao 266003, China c Qingdao Center Medical Group, Shandong, China d Center for Disease Control and Prevention, Qingdao, China

Received 8 March 2013; received in revised form 2 July 2013; accepted 9 September 2013

Abstract N-3 fatty acids (FAs) are essential FAs necessary for human health and are known to possess anticancer properties. However, the relationship between n-3 FAs and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, 4T1 mouse breast cancer cells were exposed to a representative n-3 FA, docosahexaenoic acid (DHA), to investigate the relationship between n-3 FAs and the Wnt/β-catenin signaling pathway in vivo and in vitro. In vitro studies showed that DHA strongly inhibited cell growth, and induced G1 cell cycle arrest both in 4T1 mouse breast cells and MCF-7 human breast cells. DHA reduced β-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity in 4T1 mouse breast cells. In addition, DHA down-regulated the expression of downstream target genes such as c-myc and cyclinD1. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. We found that feeding mouse the 5% fish oil-supplemented diet for 30 days significantly reduced the growth of 4T1 mouse breast cancer in vivo through inhibition of cancer cell proliferation as well as induction of apoptosis. Feeding animals a 5% fish oil diet significantly induced downregulation of β-catenin in tumor tissues with a notable increase in apoptosis. In addition, fish oil-supplemented diet decreased lung metastases of breast cancer. These observations suggested that DHA exerted its anticancer activity through down-regulation of Wnt/β-catenin signaling. Thus, our data call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer. © 2014 Elsevier Inc. All rights reserved. Keywords: Docosahexaenoic acid; Breast cancer; Fish oil; β-catenin; Wnt signaling; Tumor-bearing mouse

1. Introduction Numerous studies suggested that activation of the Wnt/β-catenin signaling pathway plays an important role in human tumor genesis [1,2]. The levels of WNTs or other components of WNT pathway are known to be altered in 50% of breast cancer cases [3]. A hallmark of the Wnt/β-catenin signaling activation is the stabilization of cytosolic β-catenin, which enters the nucleus to activate Wnt target genes by binding transcription factors of the T-cell factor/lymphoid-enhancing factor (TCF/LEF) family [4]. Mutations that activate the Wnt/β-catenin

This work was completed with the support of the Youth Science Fund project of the National Natural Science Foundation of China (81300790) and the Development project of Shandong Province medical science and technology (2013WS0262). ☆ Conflict of Interest Statement: All authors of this article have no any financial and personal relationships with other people or organisations. ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Dengzhou Roud 38#, Qingdao, Shandong Province, 266021, China. Tel.: +08-0532-82991209. E-mail address: [email protected] (M. Xue). 0955-2863/$ - see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2013.09.008

pathway generally affect β-catenin phosphorylation and stability [5]. A variety of Wnt/β-catenin target genes have been identified, including those that regulate cell proliferation and apoptosis, thus mediating cancer initiation and progression [6–8]. N-3 fatty acids (FAs) are long-chain polyunsaturated FAs. The principal dietary source of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) is from oily cold-water fish [9]. Currently, the western diet contains a disproportionally high amount of n-6 FAs and low amount of n-3 FAs, and the resulting high n-6/n-3 ratio is thought to contribute to cardiovascular disease, inflammation and cancer. Studies in human populations have linked high consumption of fish or fish oil to reduced risk of colon, prostate and breast cancer [10,11]. A number of biological effects that could contribute to cancer suppression by n-3 FAs have been suggested [12,13]. These effects include alterations in the properties of cancer cells (proliferation, invasion, metastasis and apoptosis) as well as those of host cells (inflammation, immune response and angiogenesis). Many signaling pathways are involved in these effects of n-3 FAs, including protein kinase C, ras, ERK 1/2 and NF-κB [14–17]. However, the molecular mechanisms that account for these signaling effects are not completely understood.

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To the best of our knowledge, the relationship between n-FAs and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, we sought to investigate the effect of n-FAs on β-catenin protein and protein expressions of c-myc and cyclinD1. Furthermore, the role of β-catenin in n-3 FA-mediated growth inhibition in 4T1 mouse breast cancer cells was studied in vivo. We believe that a better understanding of the mechanism of the anticancer actions of n-3 FAs would conduce to the development of new cancer therapeutic strategies involving the use of fish oil as a dietary supplement. 2. Materials and methods 2.1. Cell culture and MTT assay The 4T1 mouse breast cancer cells and MCF-7 human breast cancer cells were both purchased from Shanghai Life Science of Chinese Academy of Sciences. The cells were routinely maintained in 1:1 (v/v) mixture of DMEM high glucose (Hyclone, Beijing, China) and 10% (vol/vol) fetal bovine serum (Gibco BRL, Grand Island, NY, USA), 37°C in a tissue culture incubator with 5% CO2 and 98% relative humidity. The cells were placed in six-well plates and cultured as normal. The exponentially growing cells were used throughout the experiments. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) assay was performed as described elsewhere [18]. In brief, cells were cultured in a 96-well plate at a density of 1×105 cells per ml. The cells were then treated with 25, 50 and 100-μM DHA or EPA (Sigma Chemical Co., St. Louis, MO, USA). After 3 days, the cells were treated with 20-μl MTT (5 mg/ml). The cultures were then re-incubated for an additional 4 h. After removal of the supernatant, 150-μl DMSO was added to each well to dissolve the crystals completely, and then, the absorbance was measured at 490 nm using an ELISA Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The results are expressed as the percentage of inhibition that produced a reduction in absorbance by DHA treatment compared to the untreated controls.

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with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After three washes (5 min each) with TBST, proteins were visualized using the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Buckinghamshire, UK).The expression levels were normalized to β-actin. 2.6. Tumor growth in mice All experimental procedures were conducted in conformity with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No.8523, revised 1985). The study protocol was approved by the Review Committee for the Use of Human or Animal Subjects of Medical College of Qingdao University. Female outbreed Babl/c mice at 4 weeks of age were purchased from Shandong Laboratory Animal Center. Sixteen mice were housed in wire-top cages with sawdust bedding in a clean, air-conditioned room at a temperature of 26°C and a relative humidity of 50%. After approximately 1 week of acclimatization after arrival, the mice were randomized into two groups (8 animals per group). We injected 5×104 4T1 cells suspended in 50 μl of PBS into the mammary fat pad of each mouse. When tumors reached 8~10 mm2 in size, the mice were fed with the experimental diets. One group was treated with the control diet (containing 5% corn oil), and the other group was fed the fish oil-supplemented diet (containing 5% fish oil). These diets contained similar quantities of carbohydrates, protein, lipids, vitamins and minerals (summarized in Table 1), and the only difference is the types of lipids (i.e., corn oil vs. fish oil). Both diets were stored in sealed containers at 4°C to reduce spontaneous lipid peroxidation. Animal body weight and tumor size were measured and recorded. Tumor size was measured every 2 days in two perpendicular dimensions (a=length, b=width) with a vernier caliper and the size recorded as a volume (mm3) as calculated by a * b2/2. A tumor growth curve was then constructed, and data were presented as mean±S.E. After 30 days of tumor treatment, the mice were euthanized, and their tumors were excised and weighed. The tumor specimens were fixed in 4% formaldehyde, embedded in paraffin and cut in 4 μm sections for immunohistochemical analysis. For enumeration of pulmonary metastatic nodules, the metastases appeared as discrete white nodules on the black surface of lungs insufflated and stained with a 15% solution of India ink and then bleached by Fekette's solution. 2.7. Analysis of plasma and tissue FA levels

2.2. Apoptosis analysis The annexin V-FITC apoptosis detection kit was used for the apoptosis assay (Invitrogen, Carlsbad, CA, USA). The cells (1×106 cells/ml) were treated with 25, 50 and 100-μM DHA for 48 h. Cells were harvested by trypsinization, washed twice with PBS, and resuspended in 500 μL of binding buffer. Cell suspensions were then incubated with 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI) for 10 min at room temperature in the dark. The cells were evaluated immediately by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). 2.3. Cell cycle analysis The cells were treated with 25, 50 and 100-μM DHA for 48 h. Cells were harvested by trypsinization, washed twice with cold PBS, and then fixed with 70% cool ethanol for 2 h. After washing in cold PBS three times, cells were stained with PI (Cycle TEST PLUS DNA Reagent Kit, Becton-Dickinson, Franklin Lakes, NJ, USA). A 96-μm pore size nylonmesh was used to filter cells on the next day, and a total of 10,000 stained nuclei were analyzed with a FACScan flow cytometer with CellQuest software. 2.4. Luciferase reporter activity 4T1 cells were seeded and allowed to achieve 80% confluence in six-well plates. The cells were transiently transfected with 1 μg per well of TCF/LEF-Luc by using Lipofectamine Plus transfection reagents (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. After transfection, the cells were treated with 25, 50 and 100-μM DHA for 48 h. Cell lysates were prepared using 1× reporter lyses buffer (Promega, Madison, WI, USA). Luciferase activity was measured as described elsewhere [19], by using an AutoLumat LB953 Luminometer (Berthold, Stevenage, UK) and using the luciferase assay system from Promega. The relative luciferase activity was calculated after normalization of cellular proteins. All values are expressed as the percentage of activity relative to basal activity. 2.5. Western blot analysis 4T1 cells were seeded in six-well plates and treated with 25, 50 and 100-μM DHA. After 48 h, the cells were treated with 2% SDS (10-mM EDTA, 50-mM Tris base, 10% SDS, pH 8.0) and boiled at 100°C for 10 min. Protein concentrations were measured using the BCA protein assay. Briefly, 50-μg samples of protein were loaded on 12% SDSPAGE gels, transferred to PVDF membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) and blocked with 5% nonfat milk in TBST buffer (20-mM Tris–HCl, 120mM NaCl, 0.1% Tween for 1 h). The rabbit polyclonal antibody for β-catenin, mouse monoclonal antibody for c-myc and rabbit polyclonal antibody for cyclinD1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After three washes (5 min each) with TBST (TBS containing 0.1% Tween 20), the membranes were incubated

Plasma and breast tissues were harvested from each animal, snap-frozen in liquid nitrogen and stored in a −80°C freezer until analysis. The measurement of linoleic acid (LA), arachidonic acid (AA), EPA and DHA in plasma and tissues were conducted as described elsewhere [20]. Briefly, total lipids from plasma were extracted using the chloroform:methanol mixture (2:1, v/v), dried under a stream of nitrogen and transmethylated using boron trifluoride in methanol (14 g/L). FA methyl esters were extracted from the mixture with pentane containing 0.05% butylated hydroxytoluene. One microliter of transmethylated sample was injected into the Agilent gas chromatography 6890N linked with the 5975B mass spectrometer. Capillary column HP5-MS (30 m×0.25 mm, film thickness 0.25 μm) was used for separation and helium as the carrier gas. The column oven temperature was set at 120°C, ramped to 250°C at 3°C/min, then ramped to 300°C at 10°C/min and held at 300°C for 5 min. 2.8. Immunohistochemistry and in situ TUNEL assay for apoptotic cells Immunohistochemical analysis of β-catenin expression was performed according to the procedure described elsewhere [21]. The primary antibody is polyclonal rabbit anti-murine β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Apoptotic cell death in paraffin-embedded tumor tissue sections was examined using the TdT-FragEL

Table 1 Composition of the experimental diets used in this study

Casein (g/kg) DL-Methionine (g/kg) Sucrose (g/kg) Corn starch (g/kg) Corn oil (g/kg) Fish oil (g/kg) Cellulose (g/kg) Mineral mix, AIN-76 (170915) (g/kg) Vitamin mix, AIN-76A (40077) (g/kg) Choline bitartrate (g/kg) Ethoxyquin, antioxidant (g/kg)

Control diet

Fish oil diet

200.0 3.0 500.0 150.0 50.0

200.0 3.0 500.0 150.0

50.0 35.0 10.0 2.0 0.01

50.0 50.0 35.0 10.0 2.0 0.01

The mice were fed with the experimental diets. One group was treated with the control diet (containing 5% corn oil), and the other group was fed with the fish oilsupplemented diet (containing 5% fish oil). These diets contained similar quantities of carbohydrates, protein, lipids, vitamins and minerals, and the only difference is the types of lipids (i.e., corn oil vs. fish oil).

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Fig. 1. Inhibitory effect of DHA on the growth of 4T1 cells and MCF-7cells. (A) The 4T1 cells and MCF-7cells were treated with 25, 50 and 100-μM DHA or EPA separately for 3 days. DHA or EPA inhibited cell growth in a concentration-dependent manner (MTT assay) in 4T1 cells and MCF-7cells. Data are shown as mean±S.D. (n=5). (B) The apoptosis of 4T1 cells and MCF-7cells treated with DHA for 48 h determined by FCM. The cells were stained with annexin V-FITC and PI. The lower right quadrant (annexin V+/PI−) represents early apoptosis, and the upper right quadrant (annexin V+/PI+) represents late apoptosis and necrosis. (C) Effect of DHA on cell cycle distribution in 4T1 cells and MCF-7cells. DHA treatment resulted in a dose-dependent increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the S and G2/M phase. 1: control cells; 2–4: 4T1 cells treated with 25-, 50- and 100-μM DHA, respectively. All experiments were performed in triplicate.

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DNA fragmentation detection kit (Calbiochem, San Diego, CA, USA) according to the manufacturer's protocol. Apoptotic cells were identified as dark brown nuclei under a light microscope. The number of apoptotic cells was counted five random fields (×400) in a blinded manner.

2.9. Statistical analysis The data were expressed as mean±S.D. Statistical analysis was performed using oneway analysis of variance, followed by Duncan's test, using the statistical program, SPSS11.0 for Windows. All experiments in vitro were performed in triplicate. Differences between the control and treated groups were evaluated by Student's t test. The values obtained in the assays were considered statistically different when Pb.05.

3. Results 3.1. DHA and EPA inhibited the growth of 4T1 cells and MCF-7 cells in vitro We first determined the effect of DHA and EPA on the viability (MTT assay) of cultured 4T1 cells and MCF-7cells. As shown in Fig. 1A, treatment with DHA or EPA for 3 days inhibited the viability of 4T1 cells in a concentration-dependent manner. When 4T1 cells were treated with 25, 50 and 100-μM DHA for 3 days, we found that DHA inhibited proliferation of 4T1 cells by 25.3%, 48.9% and 79.7%, respectively. The proliferation of MCF-7 cells was also inhibited by DHA or EPA.

3.2. DHA induced apoptosis of 4T1 cells and MCF-7 cells In order to determine whether the DHA-induced reduction in cell viability was attributable to the induction of apoptosis, we estimated the numbers of apoptotic cells by flow cytometry. The proportions of apoptotic increased in a concentration-dependent manner in cells that had been treated with DHA both in 4T1 cells and MCF-7cells (Fig. 1B).

3.3. DHA altered cell cycle of 4T1 cells and MCF-7 cells The cell cycle distribution of DHA-treated and control cells were determined by flow cytometry, which gives a measure of DNA content. As shown in Fig. 1C, both in 4T1 cells and MCF-7cells, DHAtreated cells exhibited a higher proportion of cells in the G0/G1 phase as compared with control cells. On the other hand, a concomitant decrease was observed in the proportion of cells in the S/M phase relative to that observed in control cells.

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3.4. DHA down-regulated expression of β-catenin and Wnt/β-catenin target genes The effect of DHA on β-catenin protein expression was investigated by Western blot in 4T1 cells. Fig. 2A showed that 25-, 50- and 100-μM DHA treatment significantly decreased the expressive level of β-catenin protein in 4T1 cells in a dose-dependent manner. C-myc and cyclinD1 have been identified as a target of the Wnt/βcatenin signaling pathway [18]. We examined the expression of the two cellular genes. As shown in Fig. 2A, 25, 50 and 100-μM DHA treatment for 48 h resulted in significantly lower endogenous expression of c-myc and cyclinD1 than those of control group (Pb.05). Because β-catenin regulates gene expression by forming a complex with TCF/LEF transcription factor family proteins and binding to the promoter region of target genes, we further examined the effect of DHA on TCF/LEF reporter activity. TCF/LEF transcriptional activity was assayed after transient transfection of a luciferase reporter construct under the control of a TCF/LEF response element. As shown in Fig. 2B, DHA treatment significantly inhibited TCF/ LEF reporter activity in a dose-dependent fashion (Pb.05). This result strongly suggested that n-FAs modulated the Wnt/β-catenin signaling pathway. 3.5. Fish oil-supplemented diet suppressed 4T1 breast cancer growth in vivo We showed that DHA significantly inhibited 4T1 cell proliferation in vitro. Furthermore, treatment with DHA down-regulated β-catenin and its target genes expression in 4T1 cells in vitro. To further assess the anticancer efficacy of n-3 FAs in vivo, female Balb/c mice were sc inoculated with 4T1cells. After 10 days, visible tumors had developed at the injection sites (8~10 mm2 in size). Then the mice were fed with the experimental diets. One group was treated with the control diet (containing 5% corn oil), and the other group was fed with the fish oilsupplemented diet (containing 5% fish oil). As shown in Fig. 3A and Fig. 3B, after 30 days, fish oil treatment reduced the tumor volume and the tumor weight in Balb/c mice. 3.6. Fish oil-supplemented diet induced apoptosis in tumor tissue To further confirm the ability of n-3 FAs to induce apoptosis in vivo, in situ TUNEL staining was carried out on tissue sections of tumors excised on Day 30th from 4T1 cell-implanted mice fed with the experimental diets. As illustrated in Fig. 3C, fish oil treatment caused a significantly higher percentage of TUNEL-positive apoptotic

Fig. 2. The effect of DHA on Wnt/β-catenin pathway in 4T1 cells. (A) DHA down-regulated expression of β-catenin and Wnt/β-catenin target genes in 4T1 cells. Cells were treated with various concentrations of DHA for 48 h. Cell lysates were analyzed via Western blotting with the indicated antibodies. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to its own β-actin was quantified. DHA treatment significantly decreased the expressive level of β-catenin, c-myc and cyclinD1 in 4T1 cells. (B) DHA inhibited the transcriptional activity of TCF/LEF reporter in 4T1 cells. 4T1 cells were transiently transfected with p-luc TCF/LEF. After 4 h of posttransfection, the cells were incubated with 50-, 100- and 200-μM DHA, respectively, for 24 h. Luciferase activity was determined. Data are presented as mean±S.D. All experiments were performed in triplicate. 1: control cells; 2–4: 4T1 cells treated with 25-, 50- and 100-μM DHA, respectively. * Pb.05 versus the control; ** Pb.01 versus the control.

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Fig. 3. The inhibition effect of fish oil-supplemented diet on the breast cancer in vivo. Female Balb/c mice were sc inoculated with 4T1cells. When visible tumors had developed at the injection sites (8~10 mm2 in size), the mouse was fed with the experimental diets. One group was treated with the control diet (containing 5% corn oil), and the other group was fed the fish oil-supplemented diet (containing 5% fish oil). After 30 days, fish oil treatment reduced the tumor volume and the tumor weight in Balb/c mice. (A) Tumor growth curves. Each point in the curves represents the mean±S.D. (n=8 tumors). Fish oil treatment inhibited tumor growth, *Pb.05 versus control. (B) Weight of the tumors. Each bar represents the mean ±S.D. (n=8 tumors). Mean weights of the tumors are 2.43 g and 1.37 g, for the control group and fish oil treatment group, respectively. *Pb.05 versus control. (C) Fish oilsupplemented diet induced apoptosis in vivo. Representative photographs of the tumor sections examined by TUNEL assay. TUNEL-positive cell nuclei (dark brown) were observed under a fluorescence microscope (×400). The number of apoptotic cells was counted five random fields (×400) in a blinded manner. Each bar represents the “apoptosis index,” expressed as mean±S.D. Fish oil treatment caused a significantly higher percentage of TUNEL-positive apoptotic cells. *Pb.05 versus control.

Fig. 4. The effect of fish oil-supplemented diet on Wnt/β-catenin pathway in the breast cancer tissues. (A) The expression of β-catenin in excised tumors of different groups. Representative photographs of the tumor sections examined by immunohistochemical staining for β-catenin (×400). The assessment of β-catenin was based on a cytoplasm and nuclear staining pattern. Fish oil treatment caused a lower percentage of β-catenin positive cells (yellow staining in cytoplasm or brown staining in nucleus). Each bar represents the mean±S.D. (n=8 tumors).* Pb.05 versus the control. (B) Expression of β-catenin, c-myc and cyclinD1 protein in tumors detected by Western blot. Fish oil treatment resulted in significantly lower endogenous expression of β-catenin, c-myc and cyclinD1 than those of control group. (C) Fish oil-supplemented diet decreased lung metastases of breast cancer. Representative photographs of lung metastases after treatment of fish oil. Mice were euthanized 30 days after treatment. Lungs were harvested, put in a fixative (saturated picric acid: formaldehyde: acetic acid = 75:25:5) for 24 h and, then, restored the color to the lung tissue by soaking in absolute alcohol. The number of lung metastatic nodules (white) was counted. The results are presented as mean±S.D. (n=8 tumors). Compared with control mice, fish oil resulted in significantly fewer lung metastases. *Pb.05 versus control.

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cells. These results indicated that n-3 FAs could exert antitumor effect on breast cancer in vivo. 3.7. Fish oil decreased the level of β-catenin and down-regulated expression of Wnt/β-catenin target gene expression in tumor tissue The expression of β-catenin protein in excised tumors of different groups was examined by immunohistochemical staining and Western blot. As shown in Fig. 4A, the assessment of β-catenin was based on a cytoplasm and nuclear staining pattern. Fish oil treatment caused a lower percentage of β-catenin positive cells (yellow staining in cytoplasm or brown staining in nucleus). Western blot also showed that fish oil treatment resulted in a significant reduction in β-catenin expression (Pb.05) (Fig. 4B). The expression of β-catenin target genes in tumors was also detected by Western blot. Fish oil treatment resulted in significantly lower endogenous expression of c-myc and cyclinD1 than those of control group (Pb.05) (Fig. 4B). 3.8. Fish oil suppressed lung metastasis of breast cancer in vivo 4T1 cell is a highly invasive breast cancer cell line. To investigate the effect of n-FAs on invasiveness of 4T1 tumor, mice were euthanized, and the lungs were harvested for enumeration of lung metastatic nodules metastases after fish oil treatment. The results are shown in Fig. 4C. Compared with control mice and who had an average of 36 nodules per mouse, fish oil resulted in significantly fewer lung metastases (nine nodules per mouse) (Pb.05). 3.9. Concentrations of FAs in diet, plasma and tissues The concentrations of LA, AA, EPA and DHA in two different diets are shown in Table 2. The control diet mainly consisted of LA with negligible amount of AA. In comparison, the fish oil-supplemented diet mainly contained EPA and DHA, whereas the levels of LA and AA were below the detection limit. The plasma levels of essential FAs were markedly changed following the 30 days feeding of the fish oil diet. While the n-6 FA (LA and AA) concentrations were higher than the n-3 FAs (EPA and DHA) in the control diet group, it was reversed in the fish oil diet group. The animals fed with the fish oil diet had significantly increased plasma levels of DHA and EPA by 3.1- and 14.3-fold, respectively, over those of the control group. The levels of EPA and DHA in the mammary tissue were significantly increased after feeding 5% fish oil diet. 4. Discussion In this study, we found that DHA, a n-3 FA, has a strong anticancer activity in mouse breast cancer cells through a combination of multiple actions, including inhibition of cell viability and induction of

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apoptotic cell death. The strong anticancer effect was also observed in vivo in the tumor-bearing mouse. In addition, we observed a much lower degree of breast tumor metastasis in mice treated with fish oil. These results confirmed and extended earlier observation on the anticancer effect of n-3 FAs [22–24]. Various studies have shown that alterations in the β-catenin pathway may contribute to progression of breast cancer. β-catenin plays an important role as a transcriptional activator in the canonical Wnt/β-catenin signal pathway with TCF/LEF. In the absence of the Wnt protein, cytosolic β-catenin is maintained at low levels through β-catenin degradation by destruction complex machinery. Activation of Wnt signaling inhibits glycogen synthase kinase 3 beta activity and increases cytosolic β-catenin level [25,26]. The elevated β-catenin level leads to translocation of β-catenin to the nucleus and to complex formation with TCF/LEF transcription factor, which induces expression of downstream target genes that regulate cell cycle, growth and progression such as c-myc and cyclin D1 [27–30]. Increased β-catenin activity was found to be significantly correlated with the poor prognosis of breast cancer patients [31]. Consistent with these clinical data, numerous animal studies have shown that aberrant activation of the Wnt/β-catenin signalling, either by overexpression of canonical Wnt proteins or by direct stabilisation of β-catenin, can lead to mammary tumourigenesis [32,33]. In this study, we observed that n-FAs significantly reduced β-catenin levels in cytoplasm or in nucleus and suppress the transcriptional activity of Wnt/β-catenin signaling pathway in 4T1 mouse breast cells. The suppression of this transcriptional factor caused a decrease in β-catenin transcriptional activities, suppressing the expression of its target genes such as c-myc and cyclin D1 inhibiting in turn the cell proliferation. The c-myc is a potent oncogene that encodes a transcription factor, c-myc, which is of great importance in controlling cell growth and vitality [34]. C-myc protein can regulate a variety of cellular processes including cell growth and proliferation, cell-cycle progression, transcription, differentiation, apoptosis and cell motility. Cyclin D1, which forms a complex with cyclin-dependent kinase 4/6, mediates growth factor-dependent G1 phase progression and is overexpressed in a variety of cancers including human breast cancer [35,36]. The results of this study demonstrated that n-FAs downregulated the expression of c-myc and cyclinD1. The viability of 4T1 cells was efficiently inhibited, and G1 arrest in cell cycle was induced. Meanwhile, n-FAs treatment strongly presented apoptosis induction of breast cancer cell in vivo and in vitro. The 5% fish oilsupplemented diet in tumor-bearing mice reduced the tumor volume and weight. In conclusion, our results indicated that antibreast cancer activity of n-FAs is associated with its inhibitory effects on Wnt/β-catenin signaling. N-FAs caused a significant reduction in β-catenin protein level and the down-regulation of certain downstream genes in the Wnt/β-catenin pathway in breast cancer 4T1 cells in vivo and in vitro.

Table 2 The levels of n-3 FA in diets, plasma and breast tissue Diets (mg/kg diet) Control LA AA EPA DHA

37.2±6.49 / / /

Plasma (μM) Fish oil / 189±26.1 8.3±1.02 5.2±0.43

Breast (μg/g tissue)

Control

Fish oil

Control

Fish oil

227±33.6 35±4.2 ⁎⁎ 37±4.2 128±17.2

28±3.9 ⁎⁎

716±56.4 57±7.2 ⁎ 176±25.2 192±37.8

183±24.8 ⁎

149±21.6 529±64.7 ⁎⁎ 392±44.3 ⁎

493±56.2 ⁎ 725±84.6 ⁎⁎

Mice were maintained on control or fish oil diet for 30 days after the subcutaneous implantation of 4T1 cells. The control diet mainly consisted of LA with negligible amount of AA. In comparison, the fish oil-supplemented diet mainly contained EPA and DHA, whereas the levels of LA and AA were below the detection limit. The plasma levels of essential FAs were markedly changed following the 30 days feeding of the fish oil diet. While the n-6 FA (LA and AA) concentrations were higher than the n-3 FAs (EPA and DHA) in the control diet group, it was reversed in the fish oil diet group. The levels of EPA and DHA in the mammary tissue were significantly increased after feeding 5% fish oil diet. Each value is the mean±S.D. (n=8). ⁎ Pb.05 versus the control diet group. ⁎⁎ Pb.01 versus the control diet group.

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Furthermore, the antiproliferative and apoptosis induction effects of n-FAs were mediated by their ability to suppress the Wnt/β-catenin signaling, and these actions might contribute to the cancer chemoprevention efficacy of n-FAs. The results of our present study call for additional studies to determine the mechanism of n-FAs' inhibitory effects on Wnt/β-catenin signaling.

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