QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sauer, L. A. Articles by Coupland, K. Search for Related Content PubMed PubMed Citation Articles by Sauer, L. A. Articles by Coupland, K.

---

Nutrition and Cancer

Conjugated Linoleic Acid Isomers and Trans Fatty Acids Inhibit Fatty Acid Transport in Hepatoma 7288CTC and Inguinal Fat Pads in Buffalo Rats¹

Bassett Research Institute, Cooperstown, NY 13326 and ^* Centre for Lipid Research, Hull University, Hull, UK

²To whom correspondence should be addressed. E-mail: lensauermt{at}aol.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Conjugated linoleic acid (CLA) and some trans fatty acids (FA) decrease tumor growth and alter tumor and host lipid uptake and storage. The goal of this study was to test the hypothesis that the acute inhibitory effects of CLA isomers and trans FAs on FA transport in tumors and white adipose tissue are mediated via an inhibitory G-protein coupled (GPC), FFA receptor (FFAR). Experiments were performed in hepatoma 7288CTC and inguinal fat pads in Buffalo rats during perfusion in situ. CLA isomers and trans FAs (0.03–0.4 mmol/L, in plasma) were added to the arterial blood, and FA uptake or release was measured by arterial minus venous difference. In hepatoma 7288CTC, the CLA isomers, t10,c12-CLA > (±)-9-HODE [13-(S)-hydroxyoctadecadienoic acid] > t9,t11-CLA, and the trans FAs, linolelaidic = vaccenic > elaidic, decreased cAMP content and inhibited FA uptake, 13(S)-HODE release, extracellular signal-regulated kinase p44/p42 phosphorylation, and [³H]thymidine incorporation. Other CLA isomers, c9,t11-CLA, 13-(S)-HODE, c9,c11-CLA, and c11,t13-CLA, had no effect. In inguinal fat pads, FA transport was inhibited by t10,c12-CLA = linolelaidic acid > trans vaccenic acid, whereas c9,t11-CLA had no effect. In both hepatoma 7288CTC and inguinal fat pad, addition of either pertussis toxin or 8-Br-cAMP to the arterial blood reversed the inhibitions of FA transport. These results support the idea that an inhibitory GPC FFAR reduces cAMP and controls FA transport by CLA isomers and trans FAs. Ligand activity is conferred by the presence of a trans double bond proximal to the carboxyl group.

KEY WORDS: • conjugated linoleic acid • trans fatty acids • fatty acid transport • hepatoma • inguinal fat pad

The role of dietary PUFA in regulation of tumor growth has presented a conundrum in cancer research in rodents. Although these fatty acids (FA)³ may have identical carbon-chain lengths and differ only in the number, position, and configuration of the double bonds, their effects on tumor growth rates are different. Many studies have demonstrated, for example, that ingestion of dietary linoleic acid (LA; c9,c12-C18:2), an essential (n-6) FA, rather consistently increased growth rates for both transplantable, solid rodent tumors and human cancer xenografts in nude rodents (1–4). In contrast, other dietary long-chain PUFAs, including -linolenic (c9,c12,c15-C18:3) and other (n-3) FAs (5–13), as well as conjugated linoleic acid (CLA) isomers (14,15), reduced growth of transplantable rodent tumors and human cancer xenografts. Remarkably, an increased dietary LA content (9,14) reversed the reduced rate of tumor growth due to either (n-3) FAs or CLA isomers, suggesting that tumor growth may be regulated in part by interrelationships among the levels of LA, (n-3) FAs, and CLA isomers in host arterial blood. Several mechanisms have been proposed to explain the effects of LA (2–4), (n-3) FAs (10–13,16,17), and CLA isomers (14,18,19) on tumor growth; however, as yet, no definitive mechanisms are available for any FA group, and an understanding of possible interrelationships among the groups in vivo is also not clear.

In previous research, we used hepatoma 7288CTC, a fast-growing, transplantable tumor in Buffalo rats, to examine relationships among arterial blood plasma contents of LA and an (n-3) FA, eicosapentaenoic acid (EPA), on tumor LA and EPA uptakes, [³H]thymidine incorporation, and growth. Experiments were performed in vivo (2) and during perfusion in situ (3,7,11). 13-(S)-hydroxyoctadecadienoic acid (13-HODE), formed from LA by lipoxygenase activity, was identified as the mitogen responsible for LA-dependent growth in this tumor (20). The presence of either EPA or other (n-3) FAs in the arterial blood caused an acute, dose-dependent suppression of total FA and LA uptake, 13-HODE formation, and [³H]thymidine incorporation (7,11). In inguinal fat pads in normal nontumor-bearing Buffalo rats, EPA also inhibited FA uptake from the arterial blood (fed rats) and FA release into the venous blood (food-deprived rats) (11). Addition of either pertussis toxin (PTX) or 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) to the (n-3) FA-containing arterial blood reversed the suppressive effects on FA transport in both hepatoma 7288CTC and inguinal fat pads (11). We proposed that FA transport required intracellular cAMP and that a putative PTX-sensitive inhibitory G-protein coupled (GPC) (n-3) FFA receptor (FFAR) was responsible for the inhibition of adenylyl cyclase in hepatoma 7288CTC and inguinal fat pads (11,21). The decrease in formation of the mitogen, 13-HODE, caused the reduction in tumor growth (11).

Subsequent experiments indicated that t10,c12-CLA, t9,t11-CLA, and linolelaidic and elaidic acids also inhibited tumor FA uptake in hepatoma 7288CTC and FA uptake and release in inguinal fat pads in Buffalo rats during perfusion in situ (22,23). PTX and 8-Br-cAMP reversed the inhibited FA transport. A strict structural requirement for a trans double bond proximal to the carboxyl group appeared likely because c9,t11-CLA, LA, and oleic acid (c9-C18:1) did not affect FA transport. The purpose of this study was to test the hypothesis that CLA isomers and trans FAs were ligands for the putative inhibitory GPC FFAR that reduced intratumor cAMP, LA uptake, 13-HODE formation, and tumor growth. Strong support for the existence of these receptors was recently provided by the identification of a human orphan GPC FFAR (GPR 40) for which (n-3) FAs, t10,c12-CLA, and (±)-9-HODE were ligands (24,25), and GPR 41 and 43 for which short-chain FAs were ligands (26).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals, diets, and tumor implantation. Specific pathogen-free male Buffalo and Sprague-Dawley rats were purchased from Charles River Laboratories and Harlan Sprague Dawley, respectively. Rats were maintained at 23°C and 45 to 50% humidity in microisolator units (Thoren Caging Systems) in a facility approved by the Association for the Assessment and Accreditation of Laboratory Animal Care. Lighting was diurnal, 12L:12D (lights on 0600 to 1800: 300 lx), and light leaks during the dark period were excluded. Animals were given free access to water and a laboratory diet (Prolab 1000 formula, Agway). Analysis of several batches of this diet indicated that the FA content was 4.2 g FA/100 g with palmitic (21%), stearic (11%), oleic (34%), and LA (28%) as the major fatty acids. Traces of -linolenic acid were present in some batches; CLA isomers and trans FAs were not found. In some experiments, rats were food deprived for 48 h to change the direction of FA transport in inguinal fat pads. Tissue-isolated hepatomas 7288CTC were implanted in male Buffalo rats (one tumor/rat) as previously described (2,3,7,11,20,27). Tumors were used for perfusion when the estimated weights were 4 to 7 g.

    Arterial minus venous difference (A-V) measurements in hepatoma 7288CTC during perfusion in situ. Experiments were performed at 0800 to 1030 after a normal nocturnal feeding period (except for the food-deprived rats). About 50 mL donor arterial blood for perfusion were collected (0800 to 1000) from 8 Sprague-Dawley rats, either fed or food-deprived, weighing 250 to 300 g. Donor rats were anesthetized and anticoagulated, and blood was collected, filtered, and stored under mineral oil in a stirred plastic reservoir chilled in ice, as described (7,11,20,28–30).

Procedures for anesthesia and heparin treatment of the tumor-bearing rat, surgical techniques for perfusion of tissue-isolated hepatoma 7288CTC in situ, maintenance of body temperature of tumor and host rat, measurements of blood gases, and collection of arterial and venous blood samples across the tumor (2,3,7,11,20,28,29,31) and inguinal fat pad (11,29,30) were performed as previously described. The volume of the perfusion line from the reservoir to the tumor was 1.2 mL; at an arterial blood flow rate of about 0.1 mL/min, 15 min were required for the mixed reservoir blood to reach the tumor. Rates of blood flow from the tumor vein and the inguinal fat pad were 0.11 to 0.13 mL/min and 0.08 to 0.09 mL/min, respectively. Venous blood was collected passively. All surgical procedures for tumor implantation, collection of donor blood, and tumor preparation for perfusion were approved by the Institutional Animal Care and Use Committee. A diagram depicting the perfusion system is shown in Figure 1 of reference 31.

Donor blood was supplemented with 0 to 0.4 mmol/L (plasma concentration) of one of the following: c9,t11-CLA, t9,t11-CLA, t10,c12-CLA, (±)-9-HODE, 13-(S)-HODE, elaidic acid, linolelaidic acid (all from Cayman Chemical), c9,c11-CLA, c11,t13-CLA (both from Matreya), or trans vaccenic acid (Sigma Chemical). The purity of each FA was listed as 96 to 99%. GC analyses showed a single major peak and traces of 1 to 2 minor peaks, in agreement with the specifications of the supplier. To diminish the effects of carrier solvents, CLA isomers trans FAs and 13-HODE were air-dried, dissolved in arterial blood plasma, reconstituted with erythrocytes, and added to the reservoir blood, as previously described (3,7,11,20). Depending on the experiment, the donor arterial blood was also supplemented with PTX (0.5 mg/L plasma) or 10 µmol/L 8-Br-cAMP (Sigma Chemical). Labeling of the tumor DNA with [³H]thymidine and measurement of the incorporation into tumor DNA was performed as previously described (3,7,11,20,28). At the end of the experiments, the tumors were freeze-clamped and were stored at –80°C for determination of [³H]thymidine incorporation, DNA content, cAMP content, and expression of total and phosphorylated extracellular signal-regulated kinase p44/p42 (ERK1/2).

Sequential additions of CLA isomers, trans FAs, and other agents to the donor arterial blood in the reservoir were performed as previously described (11,20,28,29). CLA isomer or trans FA was added to the reservoir at 33 min to give a final plasma concentration of 0.03 to 0.2 mmol/L. At 86 min either PTX or 8-Br-cAMP was added to the arterial blood reservoir. Collection and processing of whole blood samples and tumors were as described above.

    A-V measurements in inguinal fat pads during perfusion in situ. The procedure used for perfusion of the left inguinal fat pad in situ in nontumor-bearing rats and sample collection was performed as previously described (11,29,30). Sequential additions of CLA isomers, trans FAs, PTX, or 8-Br-cAMP to the arterial blood reservoir were as described above. 13-(S)-HODE is not released by inguinal fat pads.

    Lipid extraction, analyses, and calculations. Plasma lipids, extracted from duplicate 0.2 mL of arterial and venous plasma (after addition of internal standards), were saponified and methylated, and the FAs were assayed by GC (2,3,7,11,20,28–30,32). Total FAs in arterial and venous blood represent the sum of the 7 major endogenous plasma FAs: myristic, palmitic, palmitoleic, stearic, oleic, arachidonic, and LA. Hepatoma 7288CTC has a large capacity for removal of FA from plasma triacylglycerols, phospholipids, cholesterol esters, and FFAs. Saturation of FA uptake by increases in plasma lipid content has not been observed (2,32), possibly because the increase in arterial plasma FA causes an abrupt 3- to 4-fold increase in tumor growth rate and triacylglycerol formation (27). Total FA and LA uptake in hepatoma 7288CTC and inguinal fat pads in vivo and during perfusion in situ is dependent on the rate of FA supply in the arterial blood (32). However, physiological levels of total FAs in separate batches of donor arterial blood collected from either fed or food-deprived rats often differed by as much as 20%. This variation changed the rate of the FA supply and increased the SD of calculated mean rates of FA uptake when data from similar experimental tumor and inguinal fat pad groups were pooled. Analyses showed that the percentage of supplied FA taken up by untreated tumors and fat pads was typically about 50% for tumors and 40 to 45% for fat pads, and was independent of the supply rate (29,32). Therefore, FA uptake or release rates used for statistical comparisons are reported as percentage of supply rate (defined as the FA uptake or release rate/supply rate times 100).

13-(S)-HODE was measured by HPLC as previously described (2,11,20,28). (±)-9-HODE and 13-(S)-HODE coeluted in the isocratic HPLC system used, and neither could be measured in the presence of the other. Measurable quantities of 13-(S)-HODE are not present in arterial blood plasma. Rat hepatoma 7288CTC typically converts from 1 to 5% of LA uptake to 13-HODE. Rates of 13-(S)-HODE released into the tumor venous blood, expressed as nmol/(min · g tumor), were dependent on the rate of LA supply and do not appear to be saturated at physiological LA plasma levels (2).

Plasma concentrations for CLA isomers and trans FAs were measured by GC and are given either as µmol/L or mmol/L (±1 SD). A-V measurements for CLA isomers and trans FA were converted to rates of supply and uptake as previously described (2,3,7,11,20,28–30,32) and were expressed as nmol/(min · g tumor or fat pad). Under the controlled conditions that exist during perfusion in situ, additions of exogenous CLA, trans FAs, or other agents to the arterial blood caused reproducible changes in hepatoma 7288CTC or inguinal fat pad. Because the study of 3 tumors or fat pads required killing 8 donor rats, 6 tumors or fat pads were typically examined when significant physiological effects were observed. Three tumors were examined when the added agent had no apparent physiological effect pertinent to the hypothesis tested.

    Determination of intratumor cAMP content. The cAMP content of hepatoma 7288CTC freeze-clamped in situ was measured by using Biotrak Enzyme Immunoassay System (RPN 225) and the protocol supplied by the manufacturer (Amersham-Biosciences). Portions of the tumors were pulverized in a mortar and a pestle under liquid nitrogen, and duplicate 100 mg samples were analyzed. Optical densities of samples and standards were read at 450 nm by using a Spectramax 340PC plate reader. Intratumor cAMP contents are reported as nmol/g wet weight tumor.

    Western blot measurement of tumor ERK1/2. Cytosol and membrane fractions were isolated from the frozen, pulverized tumors as described by Allgeier et al. (33). The homogenizing buffer (2 mL) contained 20 µL Halt Protease Inhibitor Cocktail (Pierce Chemical) and 10 µL Phosphatase Inhibitor Cocktails 1 and 2 (Sigma Chemical). Protein was determined by the Folin-phenol reagent (34). Cytosol preparations (27 µg total protein) were loaded and separated by 10% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane. Phosphorylated and total ERK1/2 were detected by using antibodies purchased from Promega as described in Promega’s Quick Protocol. The secondary antibody, diluted 1:5000, was Donkey Anti-Rabbit Horseradish Peroxidase purchased from Santa Cruz Biotechnology. Molecular markers (sc 2035, Santa Cruz Biotechnology), compatible with Cruz Marker Western blotting secondary antibodies, were used as internal standards. Western Blotting Kit (ECL Plus) was used for immunodetection, and bands were visualized by using Storm PhosphoImager and Image Quant software (Amersham-Biosciences).

    Statistical analysis. Results collected from analyses of individual tumors (one tumor/rat) in each treatment group are expressed as mean ± SD and were compared by one-way ANOVA with Sigma Stat 3.0.1 (Jandel Scientific). If the normality and equal variance tests were passed, multiple comparisons vs. the control group were performed by the Holm-Sidak method (35). If normality or equal variance tests failed, the Kruskal-Wallis one-way ANOVA on ranks was used, and multiple comparisons vs. the control group were performed by the Dunn’s method (35). Differences among the groups were considered statistically significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Hepatoma 7288CTC. Increased arterial blood plasma concentrations of t10,c12-CLA caused a dose-dependent reduction of the steady-state rates of total FA and LA uptakes (Fig. 1A) and 13-(S)-HODE release (Fig. 1B) in hepatoma 7288CTC perfused in situ. Complete inhibition, zero FA uptake, and 13-(S)-HODE release occurred at a plasma t10,c12-CLA concentration of about 90 µmol/L. As judged from the decrease in total FA uptake and 13-(S)-HODE release, the K_i in arterial blood plasma was 35 µmol/L. Tumor uptake of t10,c12-CLA was also dose-dependent and continued at concentrations > 90 µmol/L, even though total FA uptake was abolished (Fig. 1A, inset). Tumor [³H]thymidine incorporation (Fig. 1B) decreased from 44.8 ± 3.1 dpm/µg DNA to a plateau at about 25 dpm/µg DNA as the arterial blood plasma concentration of t10,c12-CLA was increased. The onset of the plateau in [³H]thymidine incorporation coincided with the absence of 13-(S)-HODE release. Similar low levels of [³H]thymidine incorporation were observed in vivo in hepatoma 7288CTC growing in essential FA-deficient rats (20). Plasma LA was absent in these rats, and 13-(S)-HODE was not released (20). Tumor growth in vivo in essential FA-deficient rats was dependent on dietary LA intake and tumor 13-(S)-HODE production, and [³H]thymidine incorporation during perfusion in situ was dependent on plasma LA concentrations (3,7,20). We conclude that t10,c12-CLA caused a dose-dependent decrease in [³H]thymidine incorporation, because LA uptake and 13-(S)-HODE formation became limiting.

View larger version (28K): [in this window] [in a new window]   FIGURE 1 Effects of an increase in the arterial blood plasma concentration of t10,c12-CLA on total FA and LA uptakes (A), 13-(S)-HODE release into the tumor venous blood and [3H]thymidine incorporation (B) in hepatoma 7288CTC perfused in situ. Values are means ± SD. The control value (no added t10,c12-CLA) represents results from 12 tumors. Every other data point represents results from 3 tumors. Twenty-seven tumors (one tumor/rat) were used. Rates of total FA and LA uptake and 13-HODE release were calculated from multiple A-V measurements (3 to 5 time points/tumor). Values for [3H]thymidine incorporation were duplicate determinations for each control or treated tumor. Mean tumor weight was 6.0 ± 0.4 g. The inset shows the mean (±SD) rate of tumor t10,c12-CLA uptake from the arterial blood in the 5 treatment groups. Means without a common letter differ, P < 0.05.

View larger version (33K): [in this window] [in a new window]   FIGURE 2 Effect of sequential additions of t10,c12-CLA and 8-Br-cAMP (A) or trans vaccenic acid and PTX (B) on total FA and LA uptakes and 13-(S)-HODE release in hepatoma 7288CTC perfused in situ. In each panel 3 tumors were perfused for 150 min; t10,c12-CLA or trans vaccenic acid was added at 33 min and 8-Br-cAMP (10 µmol/L plasma) or PTX (0.5 mg/L plasma) was added at 86 min. Each value at each time point is the mean ± SD for the 3 tumors. Mean tumor weight was 5.8 ± 0.2 g. Concentrations for t10,c12-CLA and trans vaccenic acid are for arterial blood plasma. Means without a common letter differ, P < 0.05.

Individual tumors in the experiments shown in Figures 1and 2 and in Table 1 each removed 51 ± 16% (n = 46) of the 7 CLA isomers and 3 trans FAs available in the arterial blood (data not shown). The inset in Figure 1A indicates that the rate of uptake of t10,c12-CLA was dependent on the rate of supply, despite the suppression of total plasma FA uptake. This result suggests that the mechanism for uptake of CLA isomers and trans FAs is dose-dependent but different from the transport mechanism for tumor uptake of plasma saturated, cis-mono-, and (n-6) cis-PUFAs. CLA isomers (t10,c12-, c9,t11-, and t9,t11-CLA) removed from the arterial blood plasma were incorporated into tumor lipids (data not shown). Mean values for pH, pO₂, and pCO₂ in untreated and CLA-containing arterial blood were 7.44 ± 0.04, 156 ± 8, and 22 ± 6, respectively, and 7.35 ± 0.02, 31 ± 12, and 52 ± 4 in tumor venous blood, respectively. The rates of arterial and tumor venous blood flow were not different among the groups. Taken together, the data indicate that the presence of the CLA isomers did not affect tumor O₂ consumption, CO₂ production, or blood flow.

The presence of either t10,c12-CLA or trans vaccenic acid in the arterial blood significantly reduced phosphorylation of ERK1/2 in hepatoma 7288CTC perfused in situ and the addition of PTX to the arterial blood restored phosphorylated ERK1/2 to control levels (Fig. 3A–D). Addition of 13-(S)-HODE to arterial blood containing t10,c12-CLA also restored phosphorylated ERK1/2 to control levels. Expression of phosphorylated ERK1/2 correlated directly with 13-HODE levels and [³H]thymidine incorporation (Table 1). Similar decreases in phosphorylated ERK1/2 and reversals by PTX were observed in tumors treated with either (±)-9-HODE or linolelaidic acid.

View larger version (61K): [in this window] [in a new window]   FIGURE 3 t10,c12-CLA or trans vaccenic acid dephosphorylated ERK1/2 in hepatomas 7288CTC perfused in situ. Each lane contained 27 µg total protein of a cytosol preparation from a single tumor perfused for 1 h. Phosphorylated ERK1/2 (p-ERK) and total ERK1/2 (ERK) were determined by Western blot analyses. Additions to the arterial blood plasma were as shown on the gel photographs. Concentrations of the additives in (A) were: lanes 1–3, none; lanes 4–6, 146 µmol/L t10,c12-CLA; lanes 7–9, 100 µmol/L t10,c12-CLA + 0.5 mg/L PTX; lanes 10–12, 109 µmol/L t10,c12-CLA + 80 µmol/L 13-(S)-HODE. Concentrations of the additives in (C) were: lanes 1–3, none; lanes 4–6, 270 µmol/L trans vaccenic acid; and lanes 7–9, 110 µmol/L trans vaccenic acid + PTX. Specific band densities (A and C) were scanned and quantified. The ratios of p-ERK to ERK are shown (B and D). Values are means ± SD (n = 3). Data points with different lower case letters are different, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

While this research was in progress, the first direct evidence for a GPC long-chain FFAR was reported (24,25); a seven-transmembrane orphan receptor (GPR 40) was identified as a partially PTX-sensitive, GPC FFAR in human heart, liver, skeletal muscle, pancreas, brain, and monocytes (24,25), and in rodent pancreatic islet cells (25). Potent activators of GPR 40 were thiazolidinedione drugs = (±)-9-HODE > (n-3) FAs > t10,c12-CLA (24) and 5,8,11-eicosatriynoic acid > (n-3) FAs > palmitic acid > elaidic acid (25). Clofibrate was inactive (24). The actions of CLA isomers containing cis double bonds proximal to the carboxyl group were not reported. Many of the human GPR 40 ligand/activators were identical to the inhibitors of FA transport reported here and elsewhere (11,21), and we tentatively concluded that the rat ortholog of human GPR 40 was the putative GPC FFAR that mediated the actions of (n-3) FAs and the inhibitors listed in Tables 1, and 2. Further support for this conclusion came from recent findings that rosiglitazone, ciglitazone, and 5,8,11,14-eicosatetraynoic acid, but not clofibrate, were potent inhibitors of intratumor cAMP content, FA uptake, and [³H]thymidine incorporation in hepatoma 7288CTC, and FA transport in inguinal fat pads and that their actions were completely reversed by either PTX or 8-Br-cAMP (unpublished results, Sauer, L. A., Dauchy, R. T., Blask, D. E., Krause, J. A., Davidson, L. K. & Dauchy, E. M., Bassett Research Institute, Cooperstown, NY). Also, we have reported that t10,c12-CLA, but not c9,t11-CLA, decreased intratumor cAMP and inhibited FA uptake, 13-(S)-HODE formation, ERK1/2 phosphorylation, and [³H]thymidine incorporation in MCF-7 human breast cancer xenografts perfused in situ in nude rats (37). Either 8-Br-cAMP or PTX reversed the inhibitory effects of t10,c12-CLA (37). Melatonin, (n-3) FAs, and t10,c12-CLA reduced FA transport and intramuscle cAMP content in hind limb skeletal muscle of Sprague-Dawley rats in vivo (38). The present list of inhibitors of FA transport now include exogenous agents: dietary (n-3) FAs, certain CLA isomers and trans FAs, thiazolidinedione drugs, and a synthetic FA; and at least 2 endogenous agents: melatonin, the neurohormone secreted by the pineal gland during darkness, and (±)-9-HODE, an oxygenated CLA produced enzymatically from LA in rodents and humans (39–41) and present in oxidized LDL (42). It will be of interest to determine if inhibition of intracellular cAMP and FA transport is an important function of (±)-9-HODE generated in vivo.

Current evidence (11,21,28,36) suggests that the steps in the signaling pathway leading to inhibition of LA-dependent growth by CLA isomers, trans and (n-3) FAs, and melatonin in hepatoma 7288CTC include the following: dose-dependent binding to an inhibitory GPC receptor (discussed above), reductions in adenylyl cyclase activity, intratumor cAMP levels, FA uptake, 13-(S)-HODE formation, phosphorylated ERK1/2, [³H]thymidine incorporation, and growth. Central unknown issues are the mechanism involved in uptake of CLA isomers, trans, and (n-3) FAs, and the mechanisms for transport and control of transport of other plasma saturated, mono-, and (n-6) PUFAs. FA transport is believed to occur either by passive diffusion [reviewed in (43)] or to require specific FA transporters [reviewed in (44)]. Both mechanisms may operate in hepatoma 7288CTC and inguinal fat pads. Either passive diffusion or a cAMP-independent transporter could mediate uptake of CLA isomers (Fig. 1) and (n-3) FAs (11,21) and trans FAs, because this uptake is dose-dependent and independent of the intracellular cAMP concentrations and the direction of transport of other plasma FAs [(11) and Fig. 1]. Transport of plasma saturated, mono-, and (n-6) PUFAs, and its control by intratumor cAMP levels almost certainly occurs via a specific cAMP-dependent FA transporter (11,21,28,36). mRNAs for the FA transporter FATP1 (fatty acid transport protein 1) are over-expressed in hepatoma 7288CTC relative to normal Buffalo rat liver (28), and we assume that it is responsible for uptake of saturated, mono-, and (n-6) PUFAs, and that its activity is controlled by cAMP. However, we have no direct evidence for this contention. FATP1 is a bifunctional protein with both FA transport and acyl-CoA synthetase activity (45). Insulin (46) and peroxisome proliferator-responsive elements (47) specific for peroxisome proliferator-activated receptor (PPAR) PPAR and PPAR are present in the promoter region of the FATP1 gene, raising the possibility that PPARs may acutely influence FATP1 activity. Insulin downregulates (46) and PPAR, (47) upregulate FATP mRNA levels. However, c9,t11-CLA was a more potent activator of human PPAR than either t10,c12-CLA or t9,t11-CLA (48), and c9,t11-CLA had no effect on FA transport in either tumors or inguinal fat pads (see Tables 1, and 2). Also clofibrate, a potent activator for PPAR, did not affect FA transport in hepatoma 7288CTC (unpublished results, Sauer, L. A., Dauchy, R. T., Blask, D. E., Krause, J. A., Davidson, L. K. & Dauchy, E. M., Bassett Research Institute, Cooperstown, NY). Both c9,t11-CLA, and t10,c12-CLA were reported to be relatively weak ligands for PPAR, in COS-1 cells transfected with expression plasmids encoding PPAR, (49). In 3T3-L1 adipocytes, t10,c12-CLA failed to activate PPAR but inhibited activation by thiazolidinedione drugs (49), suggesting possible competitive interactions. Induction of FATP1 or other proteins by PPAR agonists in vitro may require 6 to 24 h of incubation (47,48). Taken together, it seems unlikely that the rapid inhibition of FA transport caused by t10,c12-CLA and the rapid restoration of FA transport caused by t10,c12-CLA + 8-Br-cAMP (Fig. 1A) could depend on cycles of PPAR, deactivation-activation and FA transporter destruction-resynthesis. A more reasonable explanation may be kinetic control of FA transporter activity by changes in intracellular cAMP. Oligomeric complexes among FATP1 and other membrane proteins (50), if mediated by cAMP, could rapidly affect FATP1 activity. However, we are not aware of evidence that either formation of these complexes or rates of PPAR-mediated transporter synthesis are influenced by changes in intracellular cAMP levels.

A FA transporter, FAT/CD36 (fatty acid translocase) (51), is active in heart, skeletal muscle, and adipocytes but is less active in liver. In muscle, FAT/CD36 activity is acutely increased (within 30 min) during contraction and by insulin (52) through translocation of FAT/CD36 from inner cell membranes to the sarcolemma. An increase in intracellular cAMP did not affect FAT/CD36 activity in either cardiac myocytes or giant vesicles, but effects of low cAMP levels were not reported (53). Clearly, rapid kinetic effects on transporter activity may be mediated by changes in transporter location within cells (52), but it is uncertain if FAT/CD36 is present in hepatoma 7288CTC and inguinal fat pads and if decreases in intracellular cAMP levels might reduce its activity. Thus, the exact mechanism through which cAMP levels regulate FA transport activity in hepatoma 7288CTC, MCF-7 human breast cancer xenografts, rat hind limb skeletal muscle, and inguinal fat pads is not yet known. Proposed mechanisms for the steps from 13-(S)-HODE formation to increased ERK1/2 phosphorylation, [³H]thymidine incorporation, and growth were discussed in detail previously (36).

GPR 40 was recently reported in MCF-7 human breast cancer cells (54), and it is of interest to question if dietary consumption and plasma concentrations of t10,c12-CLA, trans vaccenic, linolelaidic, and elaidic acids are sufficient to influence FA transport in tumors and white adipose tissues in humans. In the Western diet, trans FAs are found in milk fat, butter fat, and ruminant meat (trans vaccenic acid and CLA isomers, mostly c9,t11-CLA) and partially hydrogenated vegetable fats and oils (linolelaidic and elaidic acids). Consumption of these trans FAs by individuals who eat the Western diet has been estimated to be 200–300 mg/d for CLA isomers (55,56), 1.5 g/d for trans vaccenic acid (57,58) and 6–8 g/d for isomeric trans FAs, including linolelaidic and elaidic acids (59). In humans ingestion of t10,c12-CLA and other CLA isomers may be too low to have significant effects (56,60), but ingestion of trans vaccenic acid as well as linolelaidic and elaidic acids at the estimated levels may raise serum levels of trans vaccenic acid and linolelaidic plus elaidic acids to 0.5% (60) and 2% of total serum FAs, respectively. As judged from the plasma LA concentration (2.3 mmol/L, 33% of total) in human subjects fed a 30% fat diet (61), the combined plasma levels of trans vaccenic, linolelaidic, and elaidic acids may be 0.2–0.3 mmol/L. Plasma levels of trans FAs in this range decreased intratumor cAMP, total and LA acid uptakes, 13-HODE release, [³H]thymidine incorporation and phosphorylated ERK1/2 in rat hepatoma 7288CTC, and FA transport in inguinal fat pads (Fig. 2 and Tables 1, and 2). Further research should determine if these trans FAs interact with GPR40 and have significant effects on tumor growth and white adipose tissue fat stores in humans.

	FOOTNOTES

 
¹ This research was supported by AICR Grant 00B037 and NCI Grant R01 CA 76197.

³ Abbreviations used: A-V, arterial minus venous difference; 8-Br-cAMP, 8-bromoadenosine-3',5'-cyclic monophosphate; CLA, conjugated linoleic acid; EPA, eicosapentaenoic acid; ERK1/2, extracellular signal-regulated kinase p44/p42; FA, fatty acids; FAT/CD36, fatty acid translocase; FATP1, fatty acid transport protein 1; FFAR, FFA receptor; GPC, G-protein coupled; (±)-9-HODE, (±)-9-hydroxyoctadecadienoic acid; 13-(S)-HODE, 13-(S)-hydroxyoctadecadienoic acid; LA, linoleic acid; PPAR, peroxisome proliferator-activated receptor; PTX, pertussis toxin.

Manuscript received 1 February 2004. Initial review completed 5 March 2004. Revision accepted 30 April 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Hillyard, L. A. & Abraham, S. (1979) Effect of dietary polyunsaturated fatty acids on growth of mammary adenocarcinomas in mice and rats. Cancer Res. 39:4430-4437.[Abstract/Free Full Text]

2. Sauer, L. A., Dauchy, R. T. & Blask, D. E. (1997) Dietary linoleic acid intake controls the arterial blood plasma concentration and the rates of growth and linoleic acid uptake and metabolism in hepatoma 7288CTC in Buffalo rats. J. Nutr. 127:1412-1421.[Abstract/Free Full Text]

3. Sauer, L. A. & Dauchy, R. T. (1998) Identification of linoleic and arachidonic acids as the factors in hyperlipemic blood that increase [³H]thymidine incorporation in hepatoma 7288CTC perfused in situ. Cancer Res. 48:3106-3111.

4. Rose, D. P., Hatala, M. A., Connolly, J. M. & Rayburn, J. (1993) Effect of diets containing different levels of linoleic acid on human breast cancer growth and lung metastasis in nude mice. Cancer Res. 53:4686-4690.[Abstract/Free Full Text]

5. Karmali, R. A., Marsh, J. & Fuchs, C. (1984) Effect of omega-3 fatty acids on growth of a rat mammary tumor. J. Natl. Cancer Inst. 73:457-461.

6. Gabor, H. & Abraham, S. (1986) Effect of dietary menhaden oil on tumor cell loss and the accumulation of mass of a transplantable mammary adenocarconoma in BALB/c mice. J. Natl. Cancer Inst. 76:1223-1229.

7. Sauer, L. A. & Dauchy, R. T. (1992) The effect of omega-3 and omega-6 fatty acids on ³H-thymidine incorporation in hepatoma 7288CTC perfused in situ. Br. J. Cancer 66:297-303.[Medline]

8. Rose, D. P. & Connolly, J. M. (1993) Effects of dietary omega-3 fatty acids on human breast cancer growth and metastasis in nude mice. J. Natl. Cancer Inst. 85:1743-1747.[Abstract/Free Full Text]

9. Hudson, E. A., Beck, S. A. & Tisdale, M. J. (1993) Kinetics of the inhibition of tumour growth in mice by eicosapentaenoic acid-reversal by linoleic acid. Biochem. Pharmacol. 45:2189-2194.[Medline]

10. Welsch, C. W., Oakley, C. S., Chang, C.-C. & Welsch, M. A. (1993) Suppression of growth by dietary fish oil of human breast carcinomas maintained in three different strains of immune-deficient mice. Nutr. Cancer 20:119-127.[Medline]

11. Sauer, L. A., Dauchy, R. T. & Blask, D. E. (2000) Mechanism for the antitumor and anticachectic effects of n-3 fatty acids. Cancer Res. 60:5289-5295.[Abstract/Free Full Text]

12. Boudreau, M. D., Sohn, K. H., Rhee, S. H., Lee, S. W., Hunt, J. D. & Hwang, D. H. (2001) Suppression of tumor cell growth both in nude mice and in culture by n-3 polyunsaturated fatty acids: mediation through cyclooxygenase-independent pathways. Cancer Res. 61:1386-1391.[Abstract/Free Full Text]

13. Sauer, L. A., Blask, D. E. & Dauchy, R. T. (2001) Mechanism of the antitumor effect of n-3 fatty acids in MCF-7 human breast cancer xenografts perfused in situ in nude rats. Proc. Am. Assoc. Cancer Res. 42:311 (abs.).

14. Cesano, A., Visonneau, S., Scimeca, J. A., Kritchevsky, D. & Santoli, D. (1998) Opposite effects of linoleic acid and conjugated linoleic acid on human prostatic cancer in SCID mice. Anticancer Res. 18:833-838.[Medline]

15. Visonneau, S., Cesano, A., Tepper, S. A., Scimeca, J. A., Santoli, D. & Kritchevsky, D. (1997) Conjugated linoleic acid suppresses the growth of human breast adenocarcinoma cells in SCID mice. Anticancer Res. 17:969-974.[Medline]

16. Palakurthi, S. S., Flckiger, R., Aktas, H., Changolkar, A. K., Shahsafaei, A., Harneit, S., Kilic, E. & Halperin, J. A. (2000) Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid. Cancer Res. 60:2919-2925.[Abstract/Free Full Text]

17. Albino, A. P., Juan, G., Traganos, F., Reinhart, L., Connolly, J., Rose, D. P. & Darzynkiewicz, Z. (2000) Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation. Cancer Res. 60:4139-4145.[Abstract/Free Full Text]

18. Banni, S., Angioni, E., Casu, V., Melis, M. P., Carta, G., Corongiu, F. P., Thompson, H. & Ip, C. (1999) Decrease in linoleic acid metabolites as a potential mechanism in cancer risk reduction by conjugated linoleic acid. Carcinogenesis 20:1019-1024.[Abstract/Free Full Text]

19. Pariza, M. W., Park, Y. & Cook, M. E. (2001) The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40:283-298.[Medline]

20. Sauer, L. A., Dauchy, R. T., Blask, D. E., Armstrong, B. J. & Scalisi, S. (1999) 13-Hydroxyoctadecadienoic acid is the mitogenic signal for linoleic acid-dependent growth in rat hepatoma 7288CTC in Vivo. Cancer Res. 59:4688-4692.[Abstract/Free Full Text]

21. Sauer, L. A., Dauchy, R. T. & Blask, D. E. (2001) Polyunsaturated fatty acids, melatonin, and cancer prevention. Biochem. Pharmacol. 61:1455-1462.[Medline]

22. Sauer, L. A., Dauchy, R. T. & Blask, D. E. (2002) Trans-10, cis-12 conjugated linoleic acid inhibits fatty acid transport in hepatoma 7288CTC and inguinal fat pads in Buffalo rats via a G_i protein-coupled signal transduction pathway. FASEB J. 16:A997 (abs.).

23. Sauer, L. A., Dauchy, R. T., Blask, D. E. & Coupland, K. (2003) Inhibition of fatty acid uptake and [³H]thymidine incorporation by conjugated linoleic acids and trans fatty acids in rat hepatoma 7288CTC: structure-function relationships. FASEB J. 17:A1155 (abs.).

24. Kotarsky, K., Nilsson, N. E., Flodgren, E., Owman, C. & Olde, B. (2003) A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs. Biochem. Biophys. Res. Commun. 301:406-410.[Medline]

25. Briscoe, C. P., Tadayyon, M., Andrews, J. L., Benson, W. G., Chambers, J. K., Eilert, M. M., Ellis, C., Eishourbagy, N. A. & Goetz, A. S., et al (2003) The orphan G protein-coupled receptor GPR 40 is activated by medium and long chain fatty acids. J. Biol. Chem. 278:11303-11311.[Abstract/Free Full Text]

26. Brown, A. J., Goldsworthy, S. M., Barnes, A. A., Eilert, M. M., Tcheang, L., Daniels, D., Muir, A. I., Wigglesworth, M. J. & Kinghorn, I., et al (2003) The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J. Biol. Chem. 278:11312-11319.[Abstract/Free Full Text]

27. Sauer, L. A., Nagel, W. O., Dauchy, R. T., Miceli, L. A. & Austin, J. E. (1986) Stimulation of tumor growth in adult rats in vivo during an acute fast. Cancer Res. 46:3469-3475.[Abstract/Free Full Text]

28. Blask, D. E., Sauer, L. A., Dauchy, R. T., Holowachuk, E. W., Ruhoff, M. S. & Kopff, H. S. (1999) Melatonin inhibition of cancer growth in vivo involves suppression of tumor fatty acid metabolism via melatonin receptor-mediated signal transduction events. Cancer Res. 59:4693-4701.[Abstract/Free Full Text]

29. Sauer, L. A., Dauchy, R. T. & Blask, D. E. (2001) Melatonin inhibits fatty acid transport in inguinal fat pads of hepatoma 7288CTC-bearing and normal Buffalo rats via receptor-mediated signal transduction. Life Sci. 68:2835-2844.[Medline]

30. Dauchy, R. T., Blask, D. E. & Sauer, L. A. (2000) Preparation of the inguinal fat pad for perfusion in situ in the rat: a surgical technique that preserves continuous blood flow. Contemp. Top. Lab. Anim. Sci. 39:29-33.[Medline]

31. Sauer, L. A. & Dauchy, R. T. (1994) Lactate release and uptake in hepatoma 7288CTC perfused in situ with L-[(U)-¹⁴C]lactate or D-[(U)-¹⁴C]glucose. Metabolism 43:1488-1497.[Medline]

32. Sauer, L. A. & Dauchy, R. T. (1992) Uptake of plasma lipids by tissue-isolated hepatomas 7288CTC and 7777 in vivo. Br. J. Cancer 66:290-296.[Medline]

33. Allgeier, A., Offermanns, S., Van Sande, J., Spicher, K., Schultz, G. & Dumont, J. E. (1994) The human thyrotropin receptor activates G-proteins G_s and G_q/11. J. Biol. Chem. 269:13733-13735.[Abstract/Free Full Text]

34. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurements with Folin-phenol reagent. J. Biol. Chem. 193:265-275.[Free Full Text]

35. Glantz, S. A. (2002) Primer of Biostatistics 5th ed. 2002 McGraw-Hill New York, NY.

36. Blask, D. E., Sauer, L. A. & Dauchy, R. T. (2002) Melatonin as a chronobiotic/anticancer agent: cellular, biochemical, and molecular mechanisms of action and their implications for circadian-based cancer therapy. Curr. Top. Med. Chem. 2:113-132.[Medline]

37. Dauchy, R. T., Dauchy, E. M., Sauer, L. A., Blask, D. E., Davidson, L. K., Krause, J. A. & Lynch, D. T. (2004) Differential inhibition of fatty acid transport in tissue-isolated steroid receptor negative human breast cancer xenografts perfused in situ with isomers of conjugated linoleic acid. Cancer Lett. 209:7-15.[Medline]

38. Dauchy, R. T., Blask, D. E., Sauer, L. A., Davidson, L. K., Krause, J. A., Smith, L. C. & Dauchy, E. M. (2004) Physiologic melatonin concentration, omega-3 fatty acids, and conjugated linoleic acid inhibit fatty acid transport in rodent hindlimb skeletal muscle in vivo. (2003)Comp. Med. 53:186-190.

39. Daret, D., Blin, P. & Larrue, J. (1989) Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets. Prostaglandins 38:203-214.[Medline]

40. Loftin, C. D. & Eling, T. E. (1996) Prostaglandin synthase 2 expression in epidermal growth factor-dependent proliferation of mouse keratinocytes. Arch. Biochem. Biophys. 330:419-429.[Medline]

41. Kawajiri, H., His, L. C., Kamitani, H., Ikawa, H., Geller, M., Ward, T., Eling, T. E. & Glasgow, W. C. (2002) Arachidonic and linoleic acid metabolism in mouse intestinal tissue: Evidence for novel lipoxygenase activity. Arch. Biochem. Biophys. 398:51-60.[Medline]

42. Nagy, L., Tontonoz, P., Alvarez, J., Chen, H. & Evans, R. M. (1998) Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR. Cell 93:229-240.[Medline]

43. Zakim, D. (1996) Fatty acids enter cells by simple diffusion. Proc. Soc. Exp. Biol. Med. 212:5-14.[Medline]

44. Fitscher, B. A., Elsing, C., Riedel, H.-D., Gorski, J. & Stremmel, W. (1996) Protein-mediated facilitated uptake processes for fatty acids, bilirubin, and other amphipathic compounds. Proc. Soc. Exp. Biol. Med. 212:15-23.[Medline]

45. Coe, N. R., Smith, A. J., Frohnert, B. I., Watkins, P. A. & Bernlohr, D. A. (1999) The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase. J. Biol. Chem. 274:36300-36304.[Abstract/Free Full Text]

46. Hui, T. Y., Frohnert, B. I., Smith, A. J., Schaffer, J. E. & Bernlohr, D. A. (1998) Characterization of the murine fatty acid transport protein gene and its insulin response sequence. J. Biol. Chem. 273:27420-27429.[Abstract/Free Full Text]

47. Frohnert, B. I., Hui, T. Y. & Bernlohr, D. A. (1999) Identification of a functional peroxisome proliferator-responsive element in the murine fatty acid transport protein gene. J. Biol. Chem. 274:3970-3977.[Abstract/Free Full Text]

48. Moya-Camarena, S. Y., Vanden Heuvel, J. P., Blanchard, S. G., Leesnitzer, L. A. & Belury, M. A. (1999) Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPAR. J. Lipid Res. 40:1426-1433.[Abstract/Free Full Text]

49. Granlund, L., Juvet, L. K., Pedersen, J. I. & Nebb, H. I. (2003) Trans10, cis12-conjugated linoleic acid prevents triacylglycerol accumulation in adipocytes by acting as a PPAR modulator. J. Lipid Res. 44:1441-1452.[Abstract/Free Full Text]

50. Richards, M. R., Listenberger, L. L., Kelly, A. A., Lewis, S. E., Ory, D. S. & Schaffer, J. E. (2003) Oligomerization of the murine fatty acid transport protein 1. J. Biol. Chem. 278:10477-10483.[Abstract/Free Full Text]

51. Brinkmann, J.F.F., Abumrad, N. A., Ibrahimi, A., van der Vusse, G. J. & Glatz, J.F.C. (2002) New insights into long-chain fatty acid uptake by heart muscle: a crucial role for fatty acid translocase/CD 36. Biochem. J. 367:561-570.[Medline]

52. Bonen, A., Luiken, J.J.F.P. & Glantz, J.F.C. (2002) Regulation of fatty acid transport and membrane transporters in health and disease. Mol. Cell. Biochem. 239:181-192.[Medline]

53. Luiken, J.J.F.P., Willems, J., Coort, S.L.M., Coumans, W. A., Bonen, A., van der Vusse, G. J. & Glatz, J.F.C. (2002) Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes. Biochem. J. 367:881-887.[Medline]

54. Yonezawa, T., Katoh, K. & Obara, Y. (2004) Existence of GPR40 in a human breast cancer cell line, MCF-7. Biochem. Biophys. Res. Commun. 314:805-809.[Medline]

55. Salminen, I., Mutanen, M., Jauhianen, M. & Aro, A. (1998) Dietary trans fatty acids increase conjugated linoleic acid levels in human serum. J. Nutr. Biochem. 9:93-98.

56. Ritzenthaler, K. L., McGuire, M. K., Falen, R., Shultz, T. D., Dasgupta, N. & McGuire, M. A. (2001) Estimation of conjugated linoleic acid intake by written dietary assessment methodologies underestimates actual intake evaluated by food duplicate methodology. J. Nutr. 131:1548-1554.[Abstract/Free Full Text]

57. Precht, D. & Molkentin, J. (1996) Rapid analysis of isomers of trans-octadecaenoic acid in milk fat. Int. Dairy J. 6:791-809.

58. Wolff, R. L. (1995) Content and distribution of trans-18:1 acids in ruminant milk and meat fats. Their importance in European diets and their effect on human milk. J. Am. Oil. Chem. Soc. 72:259-272.

59. Hunter, J. E. & Applewhite, T. H. (1986) Isomeric fatty acids in the US diet: levels and health perspectives. Am. J. Clin. Nutr. 44:707-717.[Abstract/Free Full Text]

60. Turpeinen, A. M., Mutanen, M., Aro, A., Salminen, I., Basu, S., Palmquist, D. L. & Griinari, J. M. (2002) Bioconversion of vaccenic acid to conjugated linoleic acid in humans. Am. J. Clin. Nutr. 76:504-510.[Abstract/Free Full Text]

61. Bagga, D., Ashley, J. M., Geffrey, S., Wang, H.-J., Barnard, J., Elashoff, R. & Heber, D. (1994) Modulation of serum and breast ductal fluid lipids by a very-low-fat, high-fiber diet in premenopausal women. J. Natl. Cancer Inst. 86:1419-1421.[Free Full Text]

This article has been cited by other articles:

				D. E. Blask, G. C. Brainard, R. T. Dauchy, J. P. Hanifin, L. K. Davidson, J. A. Krause, L. A. Sauer, M. A. Rivera-Bermudez, M. L. Dubocovich, S. A. Jasser, et al. Melatonin-Depleted Blood from Premenopausal Women Exposed to Light at Night Stimulates Growth of Human Breast Cancer Xenografts in Nude Rats Cancer Res., December 1, 2005; 65(23): 11174 - 11184. [Abstract] [Full Text] [PDF]

				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 Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sauer, L. A. Articles by Coupland, K. Search for Related Content PubMed PubMed Citation Articles by Sauer, L. A. Articles by Coupland, K.