Cancer Research Meeting Calendar Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, H.
Right arrow Articles by Edwards, I. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, H.
Right arrow Articles by Edwards, I. J.
[Cancer Research 65, 4442-4447, May 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Omega-3 Polyunsaturated Fatty Acids Regulate Syndecan-1 Expression in Human Breast Cancer Cells

Haiguo Sun, Isabelle M. Berquin and Iris J. Edwards

Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Requests for reprints: Iris J. Edwards, Ph.D, Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157. Phone: 336-716-2677; Fax: 33-716-6279; E-mail: iedwards{at}wfubmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human epidemiologic studies and animal model studies support a role for n-3 polyunsaturated fatty acids (n-3 PUFA) in prevention or inhibition of breast cancer. However, mechanisms for this protection remain unclear. Syndecan-1 is a heparan sulfate proteoglycan, expressed on the surface of mammary epithelial cells and known to regulate many biological processes, including cytoskeletal organization, growth factor signaling, and cell-cell adhesion. We studied effects of n-3 PUFA on syndecan-1 expression in human mammary cell lines. PUFA were delivered to cells by low-density lipoproteins (LDL) isolated from the plasma of monkeys fed diets enriched in fish oil (n-3 PUFA) or linoleic acid (n-6 PUFA). Proteoglycan synthesis was measured by incorporation of [35S]-sodium sulfate. No effect of either LDL was observed in nontumorigenic MCF-10A cells, whereas in MCF-7 breast cancer cells, treatment with n-3–enriched LDL but not n-6–enriched LDL resulted in significantly greater synthesis of a proteoglycan identified by immunoprecipitation as syndecan-1. Using real-time reverse transcription-PCR (RT-PCR), it was shown that n-3–enriched LDL significantly increased the expression of syndecan-1 mRNA in a dose-dependent manner and maximal effective time at 8 hours of treatment. The effect was mimicked by an agonist for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and eliminated by the presence of PPAR{gamma} antagonist suggesting a role for PPAR{gamma} in syndecan enhancement. Our studies show that n-3 LDL modifies the production of syndecan-1 in human breast cancer cells and suggest that biological processes regulated by syndecan-1 may be modified through LDL delivery of n-3 PUFA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human epidemiologic studies have shown that dietary intake of fish oil may protect against the development of certain cancers including breast, colon, and prostate (1–4). In support of this, animal studies have indicated that diets enriched in fish oil or its component n-3 fatty acids, docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), significantly inhibit breast cancer cell growth (5–7) and metastases (8, 9). In spite the potential importance of n-3 polyunsaturated fatty acids (PUFA) in prevention or inhibition of breast cancer, there is little understanding of the key molecular targets of n-3 PUFA that may play a role in the tumor-inhibitory properties.

Syndecan-1 (SDC-1) is a member of the syndecan family of transmembrane heparan sulfate proteoglycans and has been shown to act as a tumor suppressor molecule by induction of apoptosis and inhibition of cell growth (10–12). Expression of SDC-1 in S115 mouse mammary epithelial tumor cells was shown to restore their epithelial phenotype (13). SDC-1 expression was markedly reduced in squamous cell carcinoma and there was an inverse relationship between SDC-1 expression and tumor aggressiveness (14). Similarly, SDC-1 expression was reduced in human hepatocellular carcinoma with high metastatic potential (15) and the survival rate in patients with SDC-1–negative colorectal tumors was decreased significantly (16). Loss of tumor cell SDC-1 was associated with poor prognostic outcome in head and neck cancer (17, 18), laryngeal cancer (19), malignant mesothelioma (20), and lung cancer (21). In vitro studies have shown that SDC-1 inhibited the invasion of tumor cells into type 1 collagen (22, 23) and that expression of SDC-1 in myeloma cells suppressed matrix metalloproteinase-9 (24). Thus, decreased SDC-1 expression may be an important step in progression of carcinoma cells to the metastatic phenotype.

Factors regulating SDC-1 expression are not well understood. An intriguing finding is that the proximal promoter of the SDC-1 gene contains a functional DR-1 element (25) which is recognized by several members of the nuclear hormone superfamily receptors including peroxisome proliferator-activated receptors (PPAR). PPARs are monitors of intracellular nonesterified fatty acid level and change the transcription of many genes (26). Fatty acids are known to bind and activate PPARs (27–29). The three PPAR isotypes ({alpha}, ß/{delta}, and {gamma}) are involved in diverse physiologic processes, including cell proliferation and differentiation, apoptosis, inflammatory response, and lipid and glucose homeostasis (reviewed in refs. 30, 31). Distinct tissue distribution and developmental expression suggests distinct functions for different PPARs but clear evidence is lacking. In human breast cancer cell lines, activation of PPAR{alpha} (32) and PPARß/{delta} (33) stimulated proliferation, whereas ligands for PPAR{gamma} were growth inhibitory (34).

We have previously reported that n-3 PUFA inhibited growth and induced apoptosis in MCF-7 human breast cancer cells (35). In the present studies, we sought to determine whether SDC-1 may be a molecular target for n-3 PUFA through activation of the PPAR{gamma} transcriptional pathway and thereby provide a molecular mechanism for tumor protection by these fatty acids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of low-density lipoproteins. Low-density lipoprotein (LDL) was isolated from plasma of adult African Green monkeys fed n-3 PUFA–enriched (fish oil) or n-6 PUFA–enriched (linoleic acid) diets for 3 to 5 years and whose maintenance and clinical measurements are published (36). Blood samples drawn from the femoral vein of anesthetized animals after an overnight (18 hours) fast, were placed into chilled tubes containing 0.1% EDTA and protease inhibitor cocktail (Sigma Chemical Co., St Louis, MO) at pH 7.4. Plasma was immediately isolated by low-speed centrifugation and LDL was isolated from plasma by sequential density gradient ultracentrifugation, filter sterilized, and cryopreserved in 10% sucrose under argon at –70°C until use (37). LDL protein was measured by the method of Lowry (38). LDL chemical compositions were measured by enzymatic and chemical assays (35). LDL fatty acids were determined after lipid extraction, saponification, and methylation, and fatty acid methyl esters were measured by gas-liquid chromatography. Based on the phospholipid/protein molar ratio and the fatty acid percent composition of the LDL (35), 100 µg/mL LDL (as protein) contained ~30 µmol/L EPA and 15 µmol/L DHA.

Preparation of fatty acid-bovine serum albumin complexes. Fatty acid–free bovine serum albumin (BSA, Sigma Chemical) was prepared as a 125 µmol/L solution in DMEM/Ham's F-12. EPA, DHA, and linoleic acid, purchased as sodium salts (Sigma Chemical), were solubilized to 600 µmol/L stocks in the BSA medium and stored in aliquots at –20°C under argon (35).

Cell culture. MCF-7 and MCF-10A cell lines, obtained from American Type Culture Collection (Rockville, MD) were maintained in DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 10 mg/mL porcine insulin (Sigma Chemical), penicillin/streptomycin, and L-glutamine at 37°C in 5% CO2. In experiments measuring proteoglycan synthesis, cells were plated in 24-well plates at a density of 1.3 x 105 cells per well in growth medium. After 6 hours, the medium was changed to DMEM/F12 with 0.5% FBS for 18 hours to up-regulate LDL receptors. Proteoglycans were metabolically radiolabeled with 30 µCi/mL [35S]-sodium sulfate (Perkin-Elmer, Billerica, MA) in DMEM/F12 containing 0.5% FBS and indicated concentrations of LDL, BSA-fatty acids, or PPAR ligands troglitazone and GW259662 (Cayman Chemical, Ann Arbor, MI) for 4 to 24 hours. For experiments measuring mRNA levels, cells were plated in 6-well plates at 1 x 106 cells per well.

RNA isolation and cDNA synthesis. Total RNA was isolated using TRIZOL (Life Technologies Chemical, Ann Arbor, MI) according to manufacturer's protocol, and RNA concentrations were measured at A260. Total RNA (2 µg) was used for first-strand cDNA synthesis using Omniscript RT kit (Qiagen, Valencia, CA), Oligo (dT)12-18 Primer (Invitrogen, Carlsbad, CA), and RNase inhibitor (Promega, Madison, WI).

Real-time PCR. Real-time PCR was done using SYBR Green PCR master Mix (Applied Biosystems, Foster City, CA) on an ABI PRISM 7000 Sequence Detection System. Primers specific for SDC-1 and peptidyl prolyl isomerase B (PPIB) housekeeping gene were designed using Primer3 (39). SDC-1 primers were 5'-GGAGCAGGACTTCACCTTTG (upper) and 5'-CTCCCAGCACCTCTTTCCT (lower). PPIB primers were 5'-GCCCAAAGTCACCGTCAA (upper) and 5'-TCCGAAGAGACCAAAGATCAC (lower). PCR reactions contained 100 pmol/L of primers and 10 ng of reverse transcribed total RNA in 25 L. PCR was done with an initial 10-minute denaturation at 95°C followed by 40 cycles of PCR (15 seconds at 95°C and 1 minute at 60°C). SDC-1 data were normalized to the housekeeping control PPIB.

Proteoglycan isolation. Proteoglycans were metabolically radiolabeled with 30 µCi/mL [35S]-sodium sulfate in the presence of n-3 LDL, n-6 LDL, EPA-BSA, DHA-BSA, linoleic acid-BSA, or PPAR{delta} agonist GW610742, PPAR{gamma} agonist troglitazone, or PPAR{gamma} antagonist GW259662 plus n-3 LDL for 24 hours. Proteoglycans were isolated from the culture media (secreted proteoglycan) and cells (cell-associated proteoglycan) which were washed twice with balanced salt solution (BSS: 137 mmol/L NaCl, 2.7 mmol/L KCl, 1.45 mmol/L KH2PO4, 20.3 mmol/L Na2HPO4) and lysed on ice with 500 µL of cell lysis buffer (150 mmol/L NaCl, 25 mmol/L Tris-HCl, 1% TritonX-100, 0.1 mg/mL phenylmethylsulfonyl fluoride, 0.01 mg/mL leupeptin, 0.001 mg/mL aprotinin). Cell protein concentration was measured by the method of Lowry (38). Media and cell lysates were dialyzed exhaustively against 0.03 mol/L Na2SO4 and 0.02 mol/L NaCl at 4°C. Proteoglycans were precipitated in 1% 1-hexadecylpyridinium chloride (CPC) for 24 hours at 25°C, separated by centrifugation, and isolated by ethanol precipitation (40).

Immunoprecipitation. Radiolabeled proteoglycan were adjusted to 2.5 mg/mL BSA and incubated overnight, 4°C with 10 µg/mL polyclonal antibodies to human SDC-1 or SDC-4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 50 µL of (50% slurry) protein-A beads (Amersham Biosciences, Piscataway, NJ). Beads were pelleted by centrifugation at 10,000 x g, washed extensively (150 µmol/L NaCl, 25 mmol/L Tris-HCl, 1% Triton, 10 µg/mL pepstatin, 10 µg/mL leupeptin, 1 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride), and boiled for 2 minutes in SDS-PAGE sample buffer to release bound antigen. Bound and unbound radioactivity was measured by scintillation counting.

Data analysis. Proteoglycan synthesis was normalized by 35S dpm/µg cell protein of treatment/35S dpm/µg cell protein of control. Data were analyzed by ANOVA and Student's t test and differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of n-3 polyunsaturated fatty acids–enriched low-density lipoproteins on SDC-1 synthesis in MCF-7 and MCF-10A cells. To determine effects of n-3 PUFA on proteoglycan production, MCF-10A and MCF-7 cells were incubated in medium containing varying concentrations of n-3– or n-6–enriched LDL and [35S]-sodium sulfate. In the absence of LDL, proteoglycan synthesis was threefold higher in MCF-10A cells than that in MCF-7 cells (Fig. 1A). This confirms our recent report of significantly lower proteoglycan production in the MCF-7 compared with MCF-10A cells.1 All levels of n-3 LDL but not n-6 LDL caused a significant increase in proteoglycan synthesis, whereas no effect of either LDL was observed in MCF-10A cells. In MCF-7 cells, n-3 LDL induced a ~2-fold increase in proteoglycans.



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Effects of n-3 PUFA– and n-6 PUFA–enriched LDL on proteoglycan synthesis in MCF-7 and MCF-10A cells. Cells were preincubated in medium containing lipoprotein-deficient serum for 24 hours and with medium containing indicated concentrations of LDL and 50 µCi/mL [35S]-sodium sulfate for 24 hours. A, proteoglycans were isolated by precipitation with 1% CPC. Incorporation is normalized to total cell protein. Points, means of a single representative experiment performed in triplicate; bars, ± SE. B, isolated proteoglycans from 0 and 100 µg/mL n-3–enriched LDL cultures were immunoprecipitated by {alpha} SDC-1 or {alpha} SDC-4 antibodies. Columns, means of a single representative experiment performed in triplicate; bars, ± SE. **, P < 0.01.

 
Our previous studies have identified SDC-1 as the major species of proteoglycans produced by both of these human mammary cell lines.1 To show that the n-3 LDL specifically up-regulated SDC-1 production in the MCF-7 cells, 35S sulfate–labeled proteoglycans produced in the presence or absence of n-3 LDL were immunoprecipitated with an antibody to SDC-1. As shown in Fig. 1B, significantly more SDC-1 was measured in preparations from n-3 LDL–treated cells than control cells. SDC-4, another member of the syndecan family of proteoglycans present mammary epithelial cells did not increase when MCF-7 cells were grown in the presence of n-3 LDL (Fig. 1B).

Effects of n-3 low-density lipoproteins on SDC-1 gene expression. The observed increase in 35S sulfate incorporation was not simply a result of increased sulfation of glycosaminoglycan chains in the n-3 LDL–treated cells because double labeling with 35S sulfate and 3H serine resulted in increased incorporation of 3H serine (90.8 ± 4.6 versus 132.4 ± 19.0 dpm/µg cell protein) and 35S sulfate (8.5 ± 1.4 versus 11.0 ± 1.5 dpm/µg cell protein) for control and n-3 LDL–treated cells, respectively. Moreover, the ratio of 35S sulfate to 3H serine was similar (0.094 ± 0.014 and 0.085 ± 0.014, mean ± SE, P = 0.35) in proteoglycans from control and n-3 LDL–treated cells, indicating that the n-3 LDL–induced increase in 35S sulfate incorporation represented an increase in intact proteoglycans rather than induction of proteoglycan structural changes.

To determine whether n-3 LDL stimulation of SDC-1 production is due to transcriptional regulation of SDC-1, real-time reverse transcription-PCR (RT-PCR) was used to measure SDC-1 expression in MCF-7 cells grown in the presence or absence of n-3 LDL. Previous experiments shown in Fig. 1 had clearly shown an increase in SDC-1 protein by 24 hours of n-3 LDL treatment. To define a window of time in which LDL exerts its effects, MCF-7 cells were treated with n-3 LDL for 4, 8, 12, and 24 hours. As shown in Fig. 2A, the maximal effect was achieved by 8 hours but SDC-1 expression was still significantly higher in n-3 LDL–treated cells at 24 hours. The optimal concentration of n-3 LDL in regulating SDC-1 gene expression of was 100 µg/mL (Fig. 2B) which was also the optimal dose for proteoglycan production (Fig. 1). The marked drop in SDC-1 expression at 200 µg/mL LDL suggests toxicity of the LDL at this high concentration.



View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Time course and dose response for n-3 PUFA–enriched LDL stimulation of SDC-1 expression. A, MCF-7 cells were treated with 100 µg/mL of n-3 LDL for indicated times. Total RNA was isolated by Trizol and analyzed for SDC-1 mRNA by real-time RT-PCR. B, cells were treated with indicated concentrations of n-3 PUFA–enriched LDL for 8 hours before measurement of SDC-1 mRNA. Relative to control (=1.0). Points, means of three independent experiments performed in triplicate; bars, ±SE. **, P < 0.01; ***, P < 0.001.

 
Effect of eicosapentaenoic acid and docosahexaenoic acid on SDC-1 expression. LDL from n-3 PUFA-fed animals is enriched with two n-3 PUFA, EPA and DHA (35). To determine whether one or both of these PUFA are active in regulating the SDC-1 gene, studies were conducted with albumin-bound EPA and DHA. As shown in Fig. 3A, 30 µmol/L EPA had no effect on SDC-1 expression. This was also true for 15 and 60 µmol/L EPA (data not shown). DHA at 30 µmol/L (Fig. 3A) or 60 mol/L (data not shown) significantly (P < 0.05) increased the level of SDC-1 mRNA to a level similar to that of n-3 LDL. A combination of EPA + DHA (30 µmol/L each) did not increase the level of SDC-1 expression beyond that of DHA alone. These results suggest that regulation of SDC-1 by n-3 LDL may be primarily an effect of DHA. To confirm that the DHA-induced increase in SDC-1 mRNA resulted in an increase in SDC-1 product, a parallel study examined 35S sulfate incorporation into newly synthesized proteoglycans (Fig. 3B). Consistent with the effects on SDC-1 message levels, DHA enhanced proteoglycan synthesis to a level similar to that of n-3 LDL whereas EPA had no affect either as a separate treatment or in combination with DHA. In addition, neither n-6 LDL nor its principal component PUFA linoleic acid was effective in stimulating proteoglycan synthesis.



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Effects of EPA and DHA on SDC-1 expression. MCF-7 cells were treated with 100 µg/mL n-3 LDL, 30 µmol/L EPA-BSA, 30 µmol/L DHA-BSA, or 30 µmol/L EPA + 30 µmol/L DHA for 8 hours. A, total RNA was isolated and SDC-1 mRNA was determined by real-time RT-PCR. B, cells were treated with 100 µg/mL n-3 or n-6 LDL, 30 µmol/L EPA, DHA, EPA + DHA, or linoleic acid for 24 hours in the presence of [35S]-sodium sulfate. Proteoglycans were isolated from the medium and cells by CPC precipitation. Columns, means in triplicate experiments; bars, ±SE. Values with different subscript letters are significantly different.

 
Regulation of SDC-1 expression by peroxisome proliferator-activated receptor {gamma}. To investigate the transcriptional pathway for regulation of SDC-1 production by n-3 LDL or DHA, MCF-7 cells were incubated for 24 hours with [35S]-sodium sulfate and PPAR{gamma} agonist, troglitazone or PPAR ß/{delta} agonist, GW610742. (Fig. 4A and B). Both troglitazone and n-3 LDL were effective stimulators of proteoglycan synthesis, whereas the PPARß/{delta} agonist had no effect. The effect of troglitazone was dose dependent (B) with maximal stimulation observed at 10 µmol/L. Higher concentrations (50 µmol/L) of both troglitazone and GW610742 resulted in significant cell death (data not shown). Parallel experiments using real-time RT-PCR indicated that SDC-1 expression by 10 µmol/L troglitazone was regulated at the level of transcription (Fig. 4C). To more directly implicate PPAR{gamma} in n-3 LDL stimulation of SDC-1, cells were treated with n-3 LDL in the presence of the PPAR{gamma} antagonist GW259662. As shown in Fig. 5A, the n-3 LDL–induced increase in proteoglycan production was eliminated at all concentrations of the PPAR{gamma} antagonist. Real-time RT-PCR confirmed that the PPAR{gamma} antagonist inhibited the n-3 LDL effect on SDC-1 mRNA (Fig. 5B). This suggests that regulation of the SDC-1 gene by n-3 LDL is mediated by the PPAR{gamma} transcriptional pathway.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Effect of PPAR agonists on SDC-1 expression. A, cells were treated with 100 µg/mL n-3 or n-6 LDL or 10 µmol/L PPAR agonists for 24 hours in the presence of [35S]-sodium sulfate. Proteoglycans were isolated from the medium and cells by CPC precipitation. B, cells were treated with indicated concentrations of PPAR agonists and proteoglycan were isolated as in (A). C, cells were treated with 100 µg/mL n-3–enriched LDL or 10 µmol/L troglitazone for 8 hours. Total RNA was isolated and SDC-1 mRNA was determined by real-time RT-PCR. Columns, means of triplicate experiments; bars, ±SE. Values with different subscript letters are significantly different.

 


View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Effect of a PPAR{gamma} antagonist on n-3–enriched LDL enhancement of SDC-1. A, cells were treated with 100 µg/mL n-3 LDL ± indicated concentrations of PPAR{gamma} antagonist, GW259662 for 24 hours in the presence of [35S]-sodium sulfate. Proteoglycans were isolated from medium and cells by CPC precipitation. B, cells were treated with 100 µg/mL n-3 LDL ± 20 µmol/L GW259662 for 8 hours. Total RNA was isolated and SDC-1 mRNA was determined by real-time RT-PCR. Columns, means of experiments performed in triplicate; bars, ±SE. Values with different subscript letters are significantly different.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In spite of considerable evidence for a protective role for n-3 PUFA in breast cancer, mechanisms remain unclear. In the present studies, we report the novel finding that in human breast cancer cells, SDC-1, a multifunctional regulator of cell behavior, is a molecular target for n-3 PUFA. Expression of this proteoglycan may have important consequences in the interaction of the tumor cell and its environment.

A unique feature of these studies is the use of two delivery routes for the n-3 PUFA. Both are physiologically relevant since mammary cells in vivo receive essential fatty acids from two major sources: (a) albumin/fatty acids complexes formed following lipolysis of chylomicrons and very low density lipoproteins; (b) receptor-mediated uptake and intracellular hydrolysis of internalized LDL. The significant increase in LDL receptor activity in neoplastic tissues (41–43) suggests that the LDL pathway may be the major route for dietary fatty acids uptake by tumor cells. As shown in our previous studies, LDL delivery of n-3 PUFA has significant effects on cell growth, apoptosis and global gene regulation (35). In the present study, we have shown that at concentrations slightly lower than those found in fasted human plasma, n-3 LDL up-regulates the expression of SDC-1, that regulation is at the level of transcription and involves the PPAR{gamma} transcriptional pathway.

Interest in n-3 PUFA effects on SDC-1 was generated by the potential importance of SDC-1 in processes important to tumorigenesis (10–12, 22–24). SDC-1 is a transmembrane proteoglycan, expressed predominantly on the surface of epithelial cells. In mouse mammary epithelial cells, the SDC-1 ectodomain can bear both heparan sulfate and chondroitin sulfate glycosaminoglycan chains (44). Primarily through the heparan sulfate chains, SDC-1 interacts with extracellular matrix proteins including fibronectin (45), interstitial collagens (46), and thrombospondin (47), and with growth factors, notably fibroblast growth factor -2 (48). When the extracellular domain of SDC-1 is cross-linked by ligands, the cytoplasmic domain associates with F actin (49), suggesting that SDC-1 may be involved in extracellular-intracellular signaling. In addition, there is mounting evidence that SDC-1 is a key regulator of the migratory and invasive behavior of both normal and tumor cells (reviewed in ref. 50).

Our results showed that SDC-1 levels were significantly lower in MCF-7 breast cancer cells than in nontumorigenic MCF-10A cells. This is consistent with reduced SDC-1 in a number of malignancies (14–21) and most evidence suggests that loss of SDC-1 is directly involved in induction of the tumor cell phenotype. In mouse mammary epithelial tumor cells lacking SDC-1, epithelial morphology was restored and malignant growth properties were eliminated by re-expression of SDC-1 (13). In addition, SDC-1 synthetic analogues, termed neoglycans were shown to reduce cell viability and induce apoptosis in human myeloma and breast cancer cells (12). This highlights the importance of identifying pathways and mechanisms by which native SDC-1 production may be restored or enhanced in tumor cells.

When MCF-7 cells were grown in the presence of n-3 LDL, SDC-1 was increased up to 2-fold. The effect was associated with n-3 PUFA enrichment of the LDL, because n-6 LDL was not similarly effective. The two major long-chain n-3 PUFA in n-3–enriched LDL from these animals are EPA and DHA (35). When cells were exposed to these fatty acids bound to albumin, only DHA-BSA was able to mimic the stimulatory effect of n-3 LDL on SDC-1 production in spite of the fact that these MCF-7 cells are able to convert EPA to DHA (35). Interestingly, EPA-BSA was able to inhibit growth of MCF-7 cells although to a lesser degree than n-3 LDL (35). MCF-10A cells did not respond to the n-3 LDL by increased SDC-1. One possible explanation is that MCF-10A cell surface SDC-1 is sufficiently high that the cells are refractory to further SDC-1 induction. Another would be that some cells are more sensitive to n-3 PUFA than others.

One potential mechanism by which n-3 LDL may affect gene transcription in MCF-7 cells is by alteration in redox state. We did not measure this nor try to control it by antioxidant supplementation. The LDL was isolated from animals whose diets were supplemented with vitamins including {alpha}-tocopherol. Previous studies have shown that in LDL from similar groups of animals, the {alpha}-tocopherol remained associated with the LDL particles throughout the isolation procedure and offered protection against oxidation (51). In those studies, {alpha}-tocopherol levels were similar among the different diet groups. Nevertheless, the lipid environment did have subtle effects on in vitro oxidation of the LDL with n-6 PUFA–enriched LDL more resistant to initiation of oxidation but oxidizing at a faster rate than n-3 PUFA–enriched LDL. Clearly, effects of LDL lipids on redox state of the tumor cells would be an important area for future study.

In the present studies, both mRNA and proteoglycan were analyzed to determine effects of n-3 PUFA on SDC-1. Incorporation of both GAG and core protein precursors were increased in n-3 LDL–treated cells and proteoglycan from n-3 LDL–treated and control cells had similar ratios of core protein to GAG chains indicating that the n-3 PUFA was stimulating the production of intact proteoglycan rather than inducing structural variations in the proteoglycans. Analysis of mRNA indicated that n-3 PUFA was regulating SDC-1 expression at the level of transcription.

Our study is the first to investigate the transcriptional pathway by which n-3 PUFA regulates SDC-1 expression. Results showed that the PPAR{gamma} agonist troglitazone and n-3 LDL induced similar up-regulation of SDC-1 transcription and that the PPAR{gamma} antagonist, GW259662, was able to block the LDL induction of SDC-1. These data strongly implicate PPAR{gamma} in n-3 PUFA regulation of SDC-1. PPARs are known to monitor intracellular nonesterified fatty acid level and use fatty acids as ligands for regulating gene transcription (27, 31), but data are lacking on specific gene targets of specific fatty acids in different tissues. In a previous report, darglitazone, a ligand for both PPAR{gamma} and PPAR{delta} was shown to inhibit PUFA-stimulated expression of the proteoglycan decorin in arterial smooth muscle cells (52). Our data indicate that PPAR{gamma} agonist ligands increase rather than decrease the production of SDC-1 in MCF-7 cells. An interesting finding is that although EPA, DHA, and linoleic acid have all been shown to bind PPAR{gamma} (28), only DHA was able to regulate SDC-1 expression. This is consistent with reports that binding does not necessarily induce PPRE transactivation. Linoleic acid and arachidonic acid were both shown to bind PPAR{gamma} but did not induce transcriptional activation of PPAR{gamma}. EPA and DHA showed similar binding activity to PPAR{delta} but only EPA induced activation (28). In solving the co-crystal structure of PPAR{delta} bound to EPA, Xu et al. (27) suggested that its acyl chain length and the five cis double-bonds were important both in fitting the EPA into the PPAR pocket and in producing the numerous hydrophobic interactions required to stabilize the interaction. One speculation would be that DHA is a better fit than EPA for PPAR{gamma} although we are not aware of evidence to support this. Another possible reason for a differential result between binding and activation may be that there are indirect effects based on the fatty acids serving as precursors to more active compounds (28).

Activation of specific PPAR has been shown to result in distinct regulation of biological processes. Of great interest and relevance to the present studies are reports that in human breast cancer cell lines, including MCF-7, activation of PPAR{alpha}, and PPAR{delta}-stimulated proliferation (32, 33), whereas the PPAR{gamma} agonist troglitazone significantly inhibited growth (34). Our recent studies have shown that n-3 PUFA inhibit the growth of these cells (35). Although further investigations are needed to confirm this, we would propose, based on present studies, that the n-3 LDL–induced growth inhibition is mediated by PPAR{gamma}. Moreover, because exogenous SDC-1 ectodomain has been shown to suppress the growth of MCF-7 cells (10), we propose that the n-3 PUFA–induced inhibition of cell growth is mediated by a mechanism involving up-regulation of endogenous SDC-1 synthesis via the PPAR{gamma} transcriptional pathway.


   Acknowledgments
 
Grant support: American Institute for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
1 J.K. Kistler and I.J. Edwards. Decreased production of syndecan-1 by human breast cancer cells: implications for a role in apoptosis, submitted for publication. Back

Received 11/23/04. Revised 2/ 1/05. Accepted 3/ 1/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hursting SD, Thornquist M, Henderson MM. Types of dietary fat and the incidence of cancer at five sites. Prev Med 1990;19:242–53.[CrossRef][Medline]
  2. Sasaki S, Horacsek M, Kesteloot H. An ecological study of the relationship between dietary fat intake and breast cancer mortality. Prev Med 1993;22:187–202.[CrossRef][Medline]
  3. Caygill CP, Hill MJ. Fish, n-3 fatty acids and human colorectal and breast cancer mortality. Eur J Cancer Prev 1995;4:329–32.[Medline]
  4. Gago-Dominguez M, Yuan JM, Sun CL, Lee HP, Yu MC. Opposing effects of dietary n-3 and n-6 fatty acids on mammary carcinogenesis: the Singapore Chinese Health study. Br J Cancer 2003;89:1686–92.[CrossRef][Medline]
  5. Welsch CW, Oakley CS, Chang CC, Welsch MA. Suppression of growth by dietary fish oil of human breast carcinomas maintained in three different strains of immune-deficient mice. Nutr Cancer 1993;20:119–27.[Medline]
  6. Shao Y, Pardini L, Pardini RS. Dietary menhaden oil enhances mitomycin C antitumor activity toward human mammary carcinoma MX-1. Lipids 1995;30:1035–45.[Medline]
  7. Hardman WE, Avula CPR, Fernandes G, Cameron IL. Three percent dietary fish oil concentrate increased efficacy of doxorubicin against MDA-MB 231 breast cancer xenografts. Clin Cancer Res 2001;7:2041–9.[Abstract/Free Full Text]
  8. Rose DP, Connolly JM, Rayburn J, Coleman M. Influence of diets containing eicosapentaenoic or docosahexaenoic acid on growth and metastasis of breast cancer cells in nude mice. J Natl Cancer Inst 1995;87:587–92.[Abstract/Free Full Text]
  9. Rose DP, Connolly JM, Coleman M. Effects of {omega}-3 fatty acids on the progression of metastases after the surgical excision of human breast cancer cell solid tumors growing in nude mice. Clin Cancer Res 1996;2:1751–6.[Abstract]
  10. Mali M, Andtfolk H, Miettinen HM, Jalkanen M. Suppression of tumor cell growth by syndecan-1 ectodomain. J Biol Chem 1994;269:27795–8.[Abstract/Free Full Text]
  11. Dhodapkar MV, Abe E, Theus A, et al. Syndecan-1 is a multifunctional regulator of myeloma pathobiology: control of tumor cell survival, growth, and bone cell differentiation. Blood 1998;91:2679–88.[Abstract/Free Full Text]
  12. Pumphrey CY, Theus AM, Li S, Parrish RS, Sanderson RD. Neoglycans, carcodiimide-modified glycosaminoglycans: a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis. Cancer Res 2002;62:3722–8.[Abstract/Free Full Text]
  13. Leppä S, Mali M, Miettinen HM, Jakamen M. Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells. Proc Natl Acad Sci U S A 1992;89:932–6.[Abstract/Free Full Text]
  14. Numa F, Hirabayashi K, Kawasaki K, et al. Syndecan-1 expression in cancer of the uterine cervix: association with lymph node metastasis. Int J Oncol 2002;20:39–43.[Medline]
  15. Matsumoto A, Ono M, Fujimoto Y, Gallo RL, Bernfield M, Kohgo Y. Reduced expression of syndecan-1 in human hepatocellular carcinoma with high metastatic potential. Int J Cancer 1997;74:482–91.[CrossRef][Medline]
  16. Fujiya M, Watari J, Ashida T, et al. Reduced expression of syndecan-1 affects metastatic potential and clinical outcome in patients with colorectal cancer. Jpn J Cancer Res 2001;92:1074–81.[CrossRef][Medline]
  17. Inki P, Joensuu H, Grenman R, Klemi P, Jalkanen M. Association between syndecan-1 expression and clinical outcome in squamous cell carcinoma of the head and neck. Br J Cancer 1994;70:319–23.[Medline]
  18. Anttonen A, Kajanti M, Heikkilä P, Jalkanen M, Joensuu H. Syndecan-1 expression has prognostic significance in head and neck carcinoma. Br J Cancer 1999;79:558–64.[CrossRef][Medline]
  19. Pulkkinen JO, Penttinen M, Jalkanen M, Klemi P, Grenman R. Syndecan-1: a new prognostic marker in laryngeal cancer. Acta Otolaryngol 1997;117:312–5.[Medline]
  20. Kumar-Singh S, Jacobs W, Dhaene K, et al. Syndecan-1 expression in malignant mesothelioma: correlation with cell differentiation, WT1 expression, and clinical outcome. J Pathol 1998;186:300–5.[CrossRef][Medline]
  21. Nackaerts K, Verbeken E, Deneffe G, Vanderschueren B, Demedts M, David G. Heparan sulfate proteoglycan expression in human lung cancer cells. Int J Cancer 1997;74:335–45.[CrossRef][Medline]
  22. Liebersbach B, Sanderson RD. Expression of syndecan-1 inhibits cell invasion into type 1 collagen. J Biol Chem 1994;269:20013–9.[Abstract/Free Full Text]
  23. Kato S, Saunders S, Nguyen H, Bernfield M. Loss of cell surface syndecan-1 causes epithelia to transform into anchorage-independent mesenchyme-like cells. Mol Biol Cell 1995;6:559–76.[Abstract]
  24. Kaushal GP, Xiong X, Athota AB, Rozypal TL, Sanderson RD, Kelly T. Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9. Br J Haematol 1999;104:365–73.[CrossRef][Medline]
  25. Anisfeld AM, Kast-Woelbern HR, Meyer ME, et al. Syndecan -1 expression is regulated in an isoform specific manner by the Farnesoid-X receptor. J Biol Chem 2003;278:20420–8.[Abstract/Free Full Text]
  26. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990;347:645–50.[CrossRef][Medline]
  27. Xu HE, Lambert MH, Montana VG, et al. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol Cell 1999;3:397–403.[CrossRef][Medline]
  28. Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors {alpha} and {delta}. Proc Natl Acad Sci U S A 1997;94:4312–7.[Abstract/Free Full Text]
  29. Thoennes SR, Tate PL, Price TM, Kilgore MW. Differental transcriptional activation of peroxisome proliferator-activated receptor {gamma} by {omega}-3 and {omega}-6 fatty acids in MCF-7 cells. Mol Cell Endocrinol 2000;160:67–73.[CrossRef][Medline]
  30. Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and disease. Nature 2000;405:421–4.[CrossRef][Medline]
  31. Lee CH, Olson P, Evans RM. Lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 2003;144:2201–7.[Abstract/Free Full Text]
  32. Suchanek KM, May FJ, Robinsom JA, et al. Peroxisome proliferator-activated receptor {alpha} in the human breast cancer cell lines MCF-7 and MDA-MB-231. Mol Carcinog 2002;34:165–71.[CrossRef][Medline]
  33. Stephen RL, Gustafsson MCU, Jarvis M, et al. Activation of peroxisome proliferator-activated receptor {delta} stimulates the proliferation of human breast and prostate cancer cell lines. Cancer Res 2004;64:3162–70.[Abstract/Free Full Text]
  34. Elstner E, Müller C, Koshizuka K, et al. Ligands for peroxisome proliferator-activated receptor {gamma} and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci U S A 1998;95:8806–11.[Abstract/Free Full Text]
  35. Edwards IJ, Berquin I, Sun H, et al. Differential effects of delivery of n-3 fatty acids to human cancer cells by low density lipoproteins versus albumin. Clin Cancer Res 2004;10:8275–83.[Abstract/Free Full Text]
  36. Rudel LL, Parks JS, Hedrick CC, Thomas M, Williford K. Lipoprotein and cholesterol metabolism in diet-induced coronary artery atherosclerosis in primates. Role of cholesterol and fatty acids. Prog Lipid Res 1998;37:353–70.[CrossRef][Medline]
  37. Rumsey SC, Galeano NF, Arad Y, Deckelbaum RJ. Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins. J Lipid Res 1992;33:1551–61.[Abstract]
  38. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.[Free Full Text]
  39. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000;132:365–86.[Medline]
  40. Edwards IJ, Wagner WD. Distinct synthetic and structural characteristics of proteoglycans produced by cultured artery smooth muscle cells of atherosclerosis-susceptible pigeons. J Biol Chem 1988;263:9612–20.[Abstract/Free Full Text]
  41. Ho Y, Smith GS, Brown MS, Goldstein JL. Low density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells. Blood 1978;52:1099–104.[Free Full Text]
  42. Gal D, McDonald PC, Porter JC, Simpson ER. Cholesterol metabolism in cancer cells in monolayer culture. III. Low density lipoprotein metabolism. Int J Cancer 1981;28:315–9.[Medline]
  43. Vitols S, Peterson C, Larsson O, Holm P, Aberg B. Elevated uptake of low density lipoproteins by human lung cancer tissue in vivo. Cancer Res 1992;52:6244–7.[Abstract/Free Full Text]
  44. Rapraeger A, Jalknen M, Endo E, Koda J, Bernfield M. The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans. J Biol Chem 1985;260:11046–52.[Abstract/Free Full Text]
  45. Saunders S, Bernfield M. Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix. J Cell Biol 1988;106:423–30.[Abstract/Free Full Text]
  46. Koda JE, Rapraeger A, Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens. J Biol Chem 1985;260:8157–62.[Abstract/Free Full Text]
  47. Sun X, Mosher DF, Rapraeger A. Heparan sulfate-mediated binding of epithelial cell surface proteoglycan to thrombospondin. J Biol Chem 1989;264:2885–9.[Abstract/Free Full Text]
  48. Kiefer MC, Stephans JC, Crawford K, Okino K, Barr PJ. Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor. Proc Natl Acad Sci U S A 1990;87:6985–9.[Abstract/Free Full Text]
  49. Rapraeger A, Jalkanen M, Bernfield M. Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells. J Cell Biol 1986;103:2683–96.[Abstract/Free Full Text]
  50. Sanderson RD. Heparan sulfate proteoglycans in invasion and metastasis. Semin Cell Dev Biol 2001;12:89–98.[CrossRef][Medline]
  51. Thomas MJ, Thornburg T, Manning J, Hooper K, Rudel LL. Fatty acid composition of low density lipoprotein influences its susceptibility to autoxidation. Biochemistry 1994;33:1828–34.[CrossRef][Medline]
  52. Olsson U, Bondjers G, Camejo G. Fatty acids modulate the composition of extracellular matrix in cultured human arterial smooth muscle cells by altering the expression of genes for proteoglycan core proteins. Diabetes 1999;48:616–22.[Abstract]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
I. J. Edwards, H. Sun, Y. Hu, I. M. Berquin, J. T. O'Flaherty, J. M. Cline, L. L. Rudel, and Y. Q. Chen
In Vivo and in Vitro Regulation of Syndecan 1 in Prostate Cells by n-3 Polyunsaturated Fatty Acids
J. Biol. Chem., June 27, 2008; 283(26): 18441 - 18449.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Sun, I. M. Berquin, R. T. Owens, J. T. O'Flaherty, and I. J. Edwards
Peroxisome Proliferator-Activated Receptor {gamma}-Mediated Up-regulation of Syndecan-1 by n-3 Fatty Acids Promotes Apoptosis of Human Breast Cancer Cells
Cancer Res., April 15, 2008; 68(8): 2912 - 2919.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
C. D. Allred, D. R. Talbert, R. C. Southard, X. Wang, and M. W. Kilgore
PPAR{gamma}1 as a Molecular Target of Eicosapentaenoic Acid in Human Colon Cancer (HT-29) Cells
J. Nutr., February 1, 2008; 138(2): 250 - 256.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
V. M. Freitas, V. F. Vilas-Boas, D. C. Pimenta, V. Loureiro, M. A. Juliano, M. R. Carvalho, J. J.V. Pinheiro, A. C.M. Camargo, A. S. Moriscot, M. P. Hoffman, et al.
SIKVAV, a Laminin {alpha}1-Derived Peptide, Interacts with Integrins and Increases Protease Activity of a Human Salivary Gland Adenoid Cystic Carcinoma Cell Line through the ERK 1/2 Signaling Pathway
Am. J. Pathol., July 1, 2007; 171(1): 124 - 138.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
L. A. Sauer, R. T. Dauchy, D. E. Blask, J. A. Krause, L. K. Davidson, and E. M. Dauchy
Eicosapentaenoic Acid Suppresses Cell Proliferation in MCF-7 Human Breast Cancer Xenografts in Nude Rats via a Pertussis Toxin-Sensitive Signal Transduction Pathway
J. Nutr., September 1, 2005; 135(9): 2124 - 2129.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, H.
Right arrow Articles by Edwards, I. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, H.
Right arrow Articles by Edwards, I. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online