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Departments of Pharmacology and Therapeutics and * Chemoprevention, Roswell Park Cancer Institute, Buffalo, NY
2To whom correspondence should be addressed. E-mail: patricia.masso-welch{at}roswellpark.org.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • angiogenesis • breast • chemoprevention • conjugated linoleic acid • stroma
It has been proposed that breast cancer evolves over a long period of time, providing a challenge and an opportunity for chemopreventive intervention. In addition to changes in the mammary epithelium, changes in the stromal microenvironment were implicated in the biogenesis and progression of breast cancer. Such changes include stromal reaction induced by chronic inflammation or irradiation, resulting in fibrosis and increased angiogenesis (1–3).
One chemopreventive agent that was shown to affect the stroma is conjugated linoleic acid (CLA)3 [reviewed in (4–6)]. CLA is a minor dietary fatty acid present in meat and dairy products from ruminants; it is composed of a family of geometric and positional isomers of octadecadienoic acid [reviewed in (4–6)]. Although c9,t11-CLA is the predominant isomer present in food, lesser amounts of other CLA isomers are present as well [reviewed in (6)]. In addition to these food forms of CLA, a synthetic preparation of CLA isomers containing approximately equal amounts of the c9,t11 and t10,c12 isomers, as well as trace amounts of the other isomers, is available commercially, and was used exclusively in earlier CLA studies. Using these mixed isomer preparations, it was found that CLA inhibits mammary carcinogenesis in rats [reviewed in (6)], in part through a decrease in the density of terminal end buds (7), the primary targets of chemical carcinogens in the mammary gland (8). The ability of CLA to decrease epithelial carcinogenesis was demonstrated during initiation, promotion and progression [reviewed in (5,6)]. Metastasis of cancer cells was also shown to be inhibited by CLA (9–11).
More recently, it was demonstrated that the c9,t11 and t10,c12-CLA isomers were equally effective in inhibiting mammary carcinogenesis in rats (12) and mammary tumor metastases in mice (13). The ability of CLA to act directly on epithelial targets was shown in primary cultured mammary epithelial cells, where it was demonstrated that CLA inhibited proliferation and induced apoptosis (14).
In addition to its effects on the mammary epithelium, CLA was shown to affect the adipose stroma, by regulating the proliferation, differentiation and apoptosis of adipocytes [reviewed in (15,16)]. In addition, both the c9,t11 and t10,c12 isomers of CLA inhibit the formation of microcapillary networks by mammary stromal vascular cells in vitro, and the mixed CLA isomer preparation was shown to inhibit angiogenesis in vivo (17). The ability of mixed isomers of CLA in the diet to decrease both serum and mammary gland vascular endothelial growth factor (VEGF) concentrations in vivo (17) suggests that it may be useful in long-term chemopreventive modulation of the angiogenic environment in breast tissue.
The purpose of the current studies was to define the isomer responsible for the antiangiogenic effects of CLA in vivo. Specifically, we used the matrigel pellet angiogenesis assay to compare the efficacy of c9,t11- and t10,c12-CLA in inhibiting the formation of functional blood vessels. Serum concentrations of the proangiogenic growth factors VEGF and leptin were measured to evaluate their potential role in the antiangiogenic effects of CLA. Finally, we describe for the first time the distinct effect of c9,t11- and t10,c12-CLA isomers on the mammary adipose stroma, including the white and brown adipocytes and the existing but unstable fenestrated adipose vasculature.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Female CD2F1Cr mice were purchased from the NCI Frederick Cancer Research Facility, Biological Testing Branch (Frederick, MD), and consumed food and water ad libitum. Mice (6 wk old; n = 8–12 mice/group) were fed the semisynthetic AIN-76A–based diet supplemented with 0, 5 or 10 g/kg of c9,t11-CLA or t10,c12-CLA (Natural ASA, Hovdebygda, Norway; the purity of each isomer preparation was >90%). The remaining ingredients in the diet were 645 g/kg dextrose, 200 g/kg casein, 50 g/kg alphacel, 35 g/kg AIN-76 mineral mix, 10 g/kg AIN-76 vitamin mix, 3.0 g/kg L-methionine, 2.0 g/kg choline bitartrate and 50 g/kg corn oil (18,19). Mice were killed by cervical dislocation at the times indicated below. For the crossover studies, 3–4 mice/dietary group were fed the CLA-supplemented diets for 7 wk, followed by a reversal to the control, unsupplemented diet for 4 wk. The tumor necrosis (TNF)-
-/- (null) and TNF- +/+ (wild-type) mice were originally obtained from Dr. George Kollias at the Hellenic Pasteur Institute in Athens, Greece and are now maintained in the RPCI animal facilities. Mice from the seventh generation backcross into the C57/BL6 strain were used. TNF- wild-type (4 mice/group) or null mice (3 mice/group) were fed 0 or 1% t10,c12-CLA diets for 7 d before killing. The normal mammary gland tissues were fixed in formalin for paraffin embedding.
Animal rooms were air conditioned and humidity controlled, with a 12-h light:dark cycle. Mice were housed in accordance with the standards set by the NIH and the Roswell Park Cancer Institute Animal Care and Use Committee.
Analysis of serum VEGF and leptin concentrations.
The effects of CLA isomers on serum VEGF (VEGF-A) and leptin concentrations were assessed by ELISA using commercial kits (Quantikine M for mouse VEGF, Quantikine M for mouse leptin, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. VEGF and leptin concentrations were assessed in sera from mice after 7 wk of dietary supplementation with CLA (8 mice/group for VEGF; 12 mice/group for leptin). Sera tested for VEGF were obtained from mice that had received no additional treatment. Sera tested for leptin were obtained from mice that had received the subcutaneous matrigel challenge (see below) 1 wk before killing.
Functional angiogenesis assay.
To determine the effects of dietary CLA isomers on angiogenesis, the in vivo matrigel pellet angiogenesis assay was used as described previously (17). The matrigel pellet provides a model whereby modulatory effects on functional angiogenesis in vivo can be assessed. After 6 wk of diet treatment, mice were given subcutaneous bilateral injections of matrigel [reconstituted basement membrane from the EHS sarcoma containing 72 nmol/L (1.25 mg/L) basic fibroblast growth factor (bFGF) and 8 µmol/L (60 mg/L) heparan sulfate] into the region of the number 4 mammary gland. Mice were killed 7 d later. The pellets and adjacent normal mammary gland tissue were removed and fixed in formalin for paraffin embedding. This experiment was repeated once, with 8 mice/group in experiment A and 12 mice/group in experiment B.
Functional angiogenesis within the hematoxylin and eosin (H&E)-stained sections of the matrigel pellet was analyzed by counting the number of cell-lined structures with patent lumen and RBC in the infiltrated regions; to do this, we used an Olympus BX-40 microscope with X40 objective under epifluorescent illumination (17). All fields containing cellular-infiltrated matrigel were analyzed for the number of vessel branches containing RBC within a cell-lined vessel; areas of frank hemorrhage were excluded from analysis.
Analysis of the effects of dietary CLA on apoptosis in mammary adipose tissue.
The effect of CLA on the induction of apoptosis of the endothelial cells lining the blood vessels within the mammary fat pad was examined to determine whether the loss of brown adipose tissue (BAT) was coincident with, or secondary to the loss of the supporting vasculature. Mammary BAT was defined as regions of multilocular fat cells with a central nucleus, surrounded by an abundant supporting vasculature. Mammary white adipose tissue (WAT) was defined as regions of unilocular adipocytes with an acentric nucleus and a sparse capillary system.
Serial sections of paraffin-embedded mammary glands were prepared from mice fed control diets with or without supplementation with c9,t11-CLA or t10,c12-CLA. Paraffin sections were assayed for apoptosis by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, using the Apoptag in situ apoptosis detection kit (Intergen/Serologicals, Norcross, GA), according to the manufacturer’s instructions. TUNEL-positive blood vessels per X40 field of adipose stroma (brown fat vs. white fat) were counted using an Olympus BX40 microscope under bright field illumination, using a X40 objective (final magnification X400). Small blood vessels, which are within the same size range in cross section as small unilocular adipocytes, were distinguished by the presence of luminal RBC or morphology (small blood vessels are surrounded by stroma in addition to basement membrane, whereas adipocytes are surrounded by basement membrane alone). Capillaries were defined as vessels lined by a single cell, with signet-ring morphology when a nucleus protruding into the lumen was present in cross section. Alternately, an individual RBC may also be present in capillary cross sections. Two independent experiments were conducted. Randomly selected fields (n = 80) were counted per dietary group, with the exception of t10,c12-CLA-fed mice at d 7, for which only 28 and 27 fields for 0.5% t10,c12-CLA, and 36 and 34 fields for 1% t10,c12-CLA in experiments A and B, respectively, were available for analysis. For the mice fed t10,c12-CLA, the BAT that remained was composed largely of clusters of <5 cells; in contrast, microscope fields from the other dietary groups were composed of continuous fields of brown adipocytes.
To determine whether the effects of CLA on apoptosis in the fat pad require TNF, TNF null and TNF +/+ mice were fed the 1% t10,c12-CLA diet for 7 d, and apoptosis within the mammary fat pad was examined by TUNEL.
Analysis of mammary gland protein.
To define the effects of CLA isomers on mammary gland composition, protein concentration was determined on mammary gland lysates using the Bio-Rad DC Protein Assay Kit, based on the Lowry method (20), according to the manufacturer’s instructions. Mammary glands from mice fed control or CLA-supplemented diets were snap-frozen in liquid nitrogen and pulverized using a dry-ice cooled pulverizer. The resulting powder was weighed in a pretared, cooled weighed boat, before the addition of ice-cold lysis buffer [50 mmol/L Tris, pH 7.5, 250 mmol/L NaCl, 5 mmol/L EDTA and 0.1% (v/v) Triton X-100, containing 0.1 mmol/L phenylmethylsulfonyl fluoride, 20 µg/L leupeptin and 100 µg/L soybean trypsin inhibitor]. After 30 min of incubation on ice, the lysate was centrifuged at 13,500 x g in a Marathon 13K microfuge (Fisher Scientific) for 10 min at 4°C, and the supernatant assayed for protein concentration.
Morphologic analysis of adipocyte diameter.
H&E-stained paraffin sections were analyzed to determine the isomer-specific effects of CLA on adipocyte size. Three fields of unilocular fat per mouse were photographed, and the diameters of 15–20 adipocytes per field were measured both vertically and horizontally; these values were then averaged.
Statistics.
Data were analyzed using SigmaStat (Jandel Scientific, San Rafael, CA), using the suggested tests for the individual raw data sets. Data were evaluated by ANOVA followed by a multiple comparisons test (Tukey’s test for body weight data, Holm-Sidak Method for serum VEGF levels and Dunn’s method for serum leptin and adipocyte diameter data). The Kruskal-Wallis ANOVA ranks test was used for data from the in vivo matrigel pellet angiogenesis assay and the TUNEL assay data. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Previously, we demonstrated that mixed isomers of CLA decrease serum VEGF concentrations (17). To determine the isomer responsible for this activity, we analyzed serum concentrations of VEGF after 7 wk of continuous feeding of control or CLA-supplemented diets. Both isomers of CLA decreased serum VEGF concentration (Table 1). For c9,t11-CLA, this decrease was significant at both dietary levels; however, for t10,c12-CLA, this was significant only at the higher level (P < 0.05). In contrast, serum leptin, another angiogenic growth factor (21), was significantly decreased by both concentrations of t10,c12-CLA, but was not altered by c9,t11-CLA (Table 1).
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The mammary glands of mice fed the control diet or the 0.5% c9,t11-CLA diet were morphologically similar (Fig. 2A, B). In contrast, in mice fed the 0.5% t10,c12-CLA diet, the WAT was decreased, and the mammary BAT was virtually ablated by wk 7 in all mice examined (Fig. 2C). Interestingly, when a mixed isomer diet (17) containing approximately equal proportions of both isomers was fed, the effects were very similar to that of t10,c12-CLA alone, suggesting that in mixed preparations, the effects of the t10,c12-CLA isomer are dominant (data not shown).
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48 and 56% lower than in controls in the 0.5 and 1% t10,c12-CLA fed mice, respectively. The decreases in mammary gland weight were accompanied by 3.4- and 5.1-fold increases in protein concentration in the 0.5 and 1% diet groups, respectively. The c9,t11-CLA–fed groups did not differ from controls in mammary gland size or protein concentration. The dramatic effects of CLA on the mammary gland weight were not matched by changes in body weight. Only the 1% t10,c12-CLA diet significantly decreased body weight, and this was manifest only at 7 wk of age (Table 2).
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Preliminary experiments with mixed CLA isomers demonstrated that apoptosis was induced in the blood vessels of the fat pad, in the absence of adipocyte apoptosis, as early as 1 wk after the start of a diet supplemented with 1% CLA, demonstrating that adipose blood vessel loss precedes adipocyte death (data not shown). Therefore, early time points (d 3 and 7) were examined to determine whether the isomer-specific effects of CLA on the adipose tissue were correlated with the induction of apoptosis in the supporting adipose vasculature.
The t10,c12-CLA isomer, but not c9,t11-CLA, significantly increased the incidence of apoptosis of blood vessels within BAT within 3 d of initiation of the diet (Fig. 4). Apoptosis of blood vessels in BAT was heterogeneous, ranging from one capillary per field (Fig. 4A, arrow, left inset), to multiple branches of TUNEL-positive capillaries in several fields of residual BAT (Fig. 4A, right inset). A smaller but significant increase in the incidence of apoptosis of white adipose vasculature was induced by t10,c12- but not c9,t11-CLA (Fig. 4B). Dose-dependent effects of the t10,c12-CLA–supplemented diets on induction of apoptosis in the adipose blood vessels were seen at d 7, but not at d 3.
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We hypothesized that CLA might exert its apoptotic effects through TNF- because this cytokine was shown to induce apoptosis of brown adipocytes (24). However, t10,c12-CLA stimulated a similar robust induction of apoptosis in the adipose vasculature and the adipocytes in the mammary glands of TNF- +/+ and TNF- -/- mice (data not shown), suggesting that TNF- is not required for the apoptosis-inducing effects of t10,c12-CLA on the mammary fat pad.
Mammary unilocular adipocyte size.
Morphological effects of CLA were observed in the unilocular adipocytes of the white fat. In contrast to the isomer-specific effects of CLA on adipose blood vessel and fat cell apoptosis, both isomers of CLA significantly decreased the diameter of unilocular adipocytes of the mammary gland, an effect that was observed as early as d 3 after initiation of the diet (Table 3). After 7 d and 7 wk of continuous CLA feeding, the t10,c12 isomer induced a greater decrease in unilocular adipocyte size, which was significantly different between isomers at wk 7 (Table 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Inhibition of angiogenesis may contribute to the chemopreventive activity of CLA.
The most important observation in this study is that c9,t11-CLA and t10,c12-CLA were both effective in inhibiting angiogenesis in vivo. Both CLA isomers also decreased systemic concentrations of VEGF, a growth factor that stimulates the migration, proliferation and survival of endothelial cells [reviewed in (25)], as well as the growth and invasiveness of breast cancer cells (26). The ability of both isomers to decrease systemic VEGF may be relevant to the similar abilities of both isomers to prevent mammary cancer in rats (12) and to decrease metastatic tumor burden in mice (13).
The additional ability of the t10,c12-CLA isomer to decrease serum leptin is of interest because leptin is a proangiogenic growth factor (27) whose effects are equivalent to that of VEGF A (28). Additionally, leptin can synergize with bFGF or VEGF to induce angiogenesis (21). Our findings are in agreement with previous studies showing that mixed CLA isomers or t10,c12-CLA, but not purified c9,t11-CLA, decrease serum leptin concentrations in mice (29–31) and in genetically obese rats (32,33), as well as in humans (34,35), although not in obese men with metabolic syndrome (36).
Like VEGF, leptin can also serve as a paracrine or autocrine growth factor for breast cancer cells as well as normal mammary epithelial cells (37–39). It is important to note, however, that because leptin is derived primarily from WAT [reviewed in (40)], the t10,c12-CLA–induced decrease in serum leptin may be secondary to the loss of fat. Notably, however, a study in humans demonstrated a CLA-induced decrease in serum leptin independent of changes in body fat (34).
The ability of the t10,c12-CLA isomer to reduce leptin in addition to VEGF may confer additional anticancer activity in vivo, compared with the c9,t11-CLA isomer. In this case, a decrease in angiogenic signaling through both the VEGF and leptin pathways may result in an effective chemopreventive regimen at a lower dose. This may be an important consideration for t10,c12-CLA because emerging data suggest caution in the use of the t10,c12 isomer, at least in the obese human population where it was shown to increase insulin resistance (36).
Both c9,t11- and t10,c12-CLA decrease adipocyte size, independently of their effects on the adipose vasculature.
The decrease in adipocyte size is noteworthy because other effective chemopreventive therapies such as green tea, black tea and dietary energy restriction were shown to induce a similar decrease in the size of unilocular adipocytes (41,42). Although the relevance of this to carcinogenesis is not clear, the data suggest that that modulation of the adipose stroma may represent a previously undescribed intermediate end point common to multiple cancer chemopreventive strategies.
The mechanism whereby both isomers of CLA decrease adipocyte size may be through their ability to activate peroxisome proliferator-activated receptor (PPAR) 
[reviewed in (43)] because troglitazone, another PPAR activator, induces a similar shift to smaller adipocyte size in rats (44). Both isomers were also shown to decrease lipoprotein lipase, which would decrease lipid uptake (45). The observation that c9,t11- and t10,c12-CLA isomers differed in the extent to which they decreased adipocyte size suggests that isomer-specific mechanisms must also play a role. The c9,t11-CLA isomer induced a moderate decrease in adipocyte size that was readily reversible by a return to the control diet. Because c9,t11-CLA did not induce adipocyte apoptosis, it is possible that the induction of lipid filling of preadipocytes by the c9,t11 isomer (46,47) could result in newly differentiated, smaller adipocytes, thus decreasing the average adipocyte size in the mammary fat pad. The stimulation of lipid filling in adipocytes by the c9,t11 isomer may also contribute to the accumulation of CLA in the mammary glands, as was seen in rat studies (48). In contrast, the capacity of the t10,c12-CLA isomer to induce a more sustained decrease in adipocyte size, which was only partially reversible after a 4-wk return to a control diet, may be due to the loss of the largest adipocytes through apoptosis [(31,49) and the studies described here].
Isomer-specific effects on the adipose vasculature.
Our data are the first to demonstrate the ability of t10,c12-CLA to cause death of the adipose vasculature. This may contribute directly to the induction of adipocyte apoptosis and is likely the major reason for the decreased adipose depots in rodents fed t10,c12-CLA (31,33,50,51). Although data are contradictory concerning whether CLA causes an increase (52,53) or decrease (31,46,47,49,51,54–57) in adipocyte differentiation, the vasculature is not required for adipocyte survival in vitro. Furthermore, studies investigating the effects of CLA on preadipocytes in culture may not be relevant to the effect of dietary CLA on preexisting fat depots in the animal.
In striking contrast to the similar activity of both isomers in inhibiting de novo angiogenesis in the matrigel pellet assay in vivo (Fig. 1) and inhibiting capillary formation in vitro (17), t10,c12-CLA, but not c9,t11-CLA, induced apoptosis of the endothelium of both the brown and white mammary adipose tissue. This correlated with the ability of this isomer to induce apoptosis of the adipocytes. The capillaries of the fat, like the vasculature of all endocrine tissues, are constitutively fenestrated (21). This constitutive vascular permeability, combined with the ability of the adipose stroma and its supporting vasculature to be remodeled in the adult, resulted in the adipose vasculature being likened to that of a tumor (58). This therefore suggests that t10,c12-CLA may induce apoptosis of the tumor vasculature, potentially leading to vascular collapse and tumor regression. Interestingly, antiangiogenic agents have been shown to inhibit adipose tissue growth (58). In our studies, the induction of vascular apoptosis was specific to the adipose tissue because it was not observed in the blood vessels of the mammary gland resident lymph nodes (data not shown).
The induction of blood vessel apoptosis was especially marked in the well-vascularized BAT, whose ablation by mixed dietary CLA isomers was previously reported (6,31). Importantly, the loss of fat was not secondary to weight loss because no significant difference in weight among groups was seen at the time of maximum apoptosis, 3 and 7 d after initiation of the diets (Table 2). Indeed, only the 1% t10,c12 CLA group had a significant decrease in weight, and that was observed after 7 wk.
We propose that the abundance of well-vascularized BAT in mice may be responsible for the drastic fat loss that led to this species being described as "hyperresponsive" to the weight-reducing effects of CLA (29). The effect of this fat depletion on the ability of CLA-fed mice to support lactation is under investigation. It is anticipated that the effects on lactation in mice will be more marked than the effects on humans; women are able to lactate successfully while undergoing 1.5 g/d CLA supplementation, although the fat content of the milk is reduced (59). It is noteworthy that in larger animals in which brown fat is not abundant in the mammary gland, the BAT that is present is found in close apposition to capillaries (60) and thus is uniquely positioned to play a role in angiogenic sprouting.
Importantly, the ability of dietary t10,c12-CLA to cause rapid loss of the mouse mammary multilocular fat tissue and its accompanying well-developed vascular system may act to effectively reduce functional stromal vascular precursors in the mammary gland that are capable of responding to angiogenic challenge. However, in contrast to their isomer-specific induction of endothelial apoptosis in the blood vessels of the fat pad, c9,t,11- and t10,c12-CLA isomers acted similarly to decrease functional blood vessel recruitment in response to the injected matrigel pellet angiogenesis challenge. These results suggest that both isomers may be effective at decreasing new sprouting that occurs during angiogenesis, whereas the t10,c12 isomer may be more effective at causing apoptosis in preexisting fenestrated tumor blood vessels.
Finally, it is important to consider the relevance of CLA intake in this and other experimental studies, in comparison with the amount of CLA consumed by the human population. At a total food intake of 3.5 g/(20 g mouse · d), a 0.5% CLA-supplemented diet would supply 875 µg CLA/(g body weight · d). The efficacy of lower CLA supplementation levels at inhibiting angiogenesis has not yet been examined, but it is noteworthy that 0.1% CLA in the diet was effective at preventing carcinogenesis in rats (61). For a 275-g rat consuming 13 g/d of a diet containing 0.1% CLA, this translates into 47 µg/g body weight of CLA consumed daily. Using an FFQ, Aro et al. (62) and Voorrips et al. (63) reported that women in the highest quintile of CLA intake consumed 204 and 290 mg/d, respectively. Using the former value, a 64-kg woman would consume 3 µg CLA/(g body weight · d). Actual values are probably higher because FFQ may underestimate CLA intake (64). Moreover, there is substantial conversion of vaccenic acid (trans-11 C18:1) from dairy fat to CLA in tissues (65). Thus, the quantitation of both vaccenic acid and CLA in the human diet may provide a more appropriate comparison of actual intake in humans vs. the amount of synthetic CLA fed in experimental studies reported herein. Nevertheless, it is likely that CLA-enriched functional foods or supplementation will be required for maximal benefit. In this regard, CLA supplements as high as 6.8 g/d [106 µg CLA/(g body weight · d) for a 64-kg woman] are currently being tested clinically in obese and diabetic subjects (66).
A model was developed to summarize the mechanisms whereby c9,t11- and t10,c12-CLA isomers differ in their effects on angiogenesis as well as the survival of preexisting mammary vasculature (Fig. 5). Inhibition of angiogenesis through systemic decreases in serum VEGF by both isomers may contribute to their similar activities in inhibiting angiogenesis in vivo in the matrigel pellet, through decreasing the availability, motility or invasion of local endothelial progenitors, their morphogenesis into lumen-containing blood vessels and connection to preexisting functional blood vessels, and/or maintenance of their patency and survival after formation. Regardless of the mechanism, the end result is a predominance of solid cords of cells rather than functional patent vascular tubules. Furthermore, the mammary gland may serve as a reservoir for long-acting effects of c9,t11-CLA; however the adipose-reducing qualities of t10,c12-CLA in mice suggest that it is only the c9,t11 isomer of CLA that is likely to accumulate appreciably in the mouse mammary gland. The additional isomer-specific ability of t10,c12-CLA to decrease leptin and disrupt the preexisting fenestrated vasculature of adipose tissue suggests that it may be useful therapeutically to interrupt the similar vasculature of preexisting tumors, as well as in prevention strategies for women at high risk for recurrence of occult breast cancer disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BAT, brown adipose tissue; bFGF, basic fibroblast growth factor; CLA, conjugated linoleic acid; H&E, hematoxylin and eosin; PPAR, peroxisome proliferator-activated receptor; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; VEGF, vascular endothelial growth factor; WAT, white adipose tissue.
Manuscript received 22 August 2003. Initial review completed 22 September 2003. Revision accepted 21 October 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Ohtani, H. (1998) Stromal reaction in cancer tissue: pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions. Pathol. Intern. 48:1-9.[Medline]
2. Dvorak, H. F. (1986) Tumors: wounds that do not heal. N. Engl. J. Med. 315:1650-1659.[Medline]
3. Kessler, D. A., Langer, R. S., Pless, N. A. & Folkman, J. (1976) Mast cells and tumor angiogenesis. Int. J. Cancer 18:703-709.[Medline]
4. Pariza, M. W., Park, Y. & Cook, M. E. (2001) The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40:283-298.[Medline]
5. Belury, M. A. (2002) Inhibition of carcinogenesis by conjugated linoleic acid: potential mechanisms of action. J. Nutr. 132:2995-2998.
6. Ip, M. M., Masso-Welch, P. A. & Ip, C. (2003) Prevention of mammary cancer with conjugated linoleic acid: role of the stroma and the epithelium. J. Mamm. Gland Biol. Neoplasia 8:103-118.[Medline]
7. Thompson, H., Zhu, Z. J., Banni, S., Darcy, K., Loftus, T. & Ip, C. (1997) Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk. Cancer Res 57:5067-5072.
8. Russo, I. H. & Russo, J. (1978) Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst. 61:1439-1449.
9. 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 18:1429-1434.[Medline]
10. Hubbard, N. E., Lim, D., Summers, L. & Erickson, K. L. (2000) Reduction of murine mammary tumor metastasis by conjugated linoleic acid. Cancer Lett 150:93-100.[Medline]
11. 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 17:969-973.[Medline]
12. Ip, C., Dong, Y., Ip, M. M., Banni, S., Carta, G., Angioni, E., Murro, E., Spada, S., Melis, M. P. & Saebo, A. (2002) Conjugated linoleic acid isomers and mammary cancer prevention. Nutr. Cancer 43:52-58.[Medline]
13. Hubbard, N. E., Lim, D. & Erickson, K. L. (2003) Effect of separate conjugated linoleic acid isomers on murine mammary tumorigenesis. Cancer Lett 190:13-19.[Medline]
14. Ip, M. M., Masso-Welch, P. A., Shoemaker, S. F., Shea-Eaton, W. K. & Ip, C. (1999) Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture. Exp. Cell Res. 250:22-34.[Medline]
15. Belury, M. A. (2002) Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu. Rev. Nutr. 22:505-531.[Medline]
16. Evans, M. E., Brown, J. M. & McIntosh, M. K. (2003) Isomer-specific effects of conjugated linoleic acid (CLA) on adiposity and lipid metabolism. J. Nutr. Biochem. 13:508-516.
17. Masso-Welch, P. A., Zangani, D., Ip, C., Vaughan, M. M., Shoemaker, S. F., Ramirez, R. A. & Ip, M. M. (2002) Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid. Cancer Res 62:4383-4389.
18. American Institute of Nutrition (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.
19. American Institute of Nutrition (1980) Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 110:1726.
20. Grant, K. I. (1990) LOWRY—a simple BASIC program for the analysis of protein assays over a wide range of concentrations. Comput. Methods Programs Biomed. 31:237-242.[Medline]
21. Cao, R., Brakenhielm, E., Wahlestedt, C., Thyberg, J. & Cao, Y. (2001) Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc. Natl. Acad. Sci. U.S.A. 98:6390-6395.
22. Trayhurn, P. & Beattie, J. H. (2001) Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proc. Nutr. Soc. 60:329-339.[Medline]
23. Chen, H. C. & Farese, R. V., Jr (2001) Turning WAT into BAT gets rid of fat. Nat. Med. 7:1102-1103.[Medline]
24. Nisoli, E., Briscini, L., Giordano, A., Tonello, C., Wiesbrock, S. M., Uysal, K. T., Cinti, S., Carruba, M. O. & Hotamisligil, G. S. (2000) Tumor necrosis factor alpha mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity. Proc. Natl. Acad. Sci. U.S.A. 97:8033-8038.
25. Robinson, C. J. & Stringer, S. E. (2001) The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J. Cell. Sci. 114:853-865.[Abstract]
26. Price, D. J., Miralem, T., Jiang, S., Steinberg, R. & Avraham, H. (2001) Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells. Cell Growth Differ 12:129-135.
27. Sierra-Honigmann, M. R., Nath, A. K., Murakami, C., Garcia-Cardena, G., Papapetropoulos, A., Sessa, W. C., Madge, L. A., Schechner, J. S., Scwabb, M. B., Polverini, P. J. & Flores-Riveros, J. R. (1998) Biological action of leptin as an angiogenic factor. Science (Washington, DC) 281:1683-1686.
28. Bouloumie, A., Drexler, H.C.A., Lafontan, M. & Busse, R. (1998) Leptin, the product of Ob gene, promotes angiogenesis. Circ. Res. 83:1059-1066.
29. Akahoshi, A., Goto, Y., Murao, K., Miyazaki, T., Yamasaki, M., Nonaka, M., Yamada, K. & Sugano, M. (2002) Conjugated linoleic acid reduces body fats and cytokine levels of mice. Biosci. Biotechnol. Biochem. 66:916-920.[Medline]
30. Clement, L., Poirier, H., Niot, I., Bocher, V., Guerre-Millo, M., Krief, S., Staels, B. & Besnard, P. (2002) Dietary trans-10, cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse. J. Lipid Res. 43:1400-1409.
31. Tsuboyama-Kasaoka, N., Takahashi, N., Tanemura, K., Kim, H.-J., Tange, T., Okuyama, H., Kasai, M., Ikemoto, S. & Ezaki, O. (2000) Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes 49:1534-1542.[Abstract]
32. Rahman, S. M., Wang, Y.-M., Yotsumoto, H., Cha, J.-Y., Han, S.-Y., Inoue, S. & Yanagita, T. (2001) Effects of conjugated linoleic acid on serum leptin concentration, body-fat accumulation, and ß-oxidation of fatty acid in OLETF rats. Nutrition 17:385-390.[Medline]
33. Ryder, J. W., Portocarrero, C. P., Song, X. M., Cui, L., Yu, M., Combatsiaris, T., Galuska, D., Bauman, D. E., Barbano, D. M., Charron, M. J., Zierath, J. R. & Houseknecht, K. L. (2001) Isomer-specific antidiabetic properties of conjugated linoleic acid. Diabetes 50:1149-1157.
34. Medina, E., Horn, W., Keim, N., Havel, P., Benito, P., Kelley, D., Nelson, G. & Erickson, K. (2000) Conjugated linoleic acid supplementation in humans: effects on circulating leptin concentrations and appetite. Lipids 35:783-788.[Medline]
35. Belury, M. A., Mahon, A. & Banni, S. (2003) The conjugated linoleic acid (CLA) isomer, t10c12-CLA, is inversely associated with changes in body weight and serum leptin in subjects with type 2 diabetes mellitus. J. Nutr. 133:257S-260S.
36. Riserus, U., Arner, P., Brismar, K. & Vessby, B. (2002) Treatment with dietary trans10cis12 conjugated linoleic acid causes isomer-specific insulin resistance in obese men with the metabolic syndrome. Diabetes Care 25:1516-1521.
37. O’Brien, S. N., Welter, B. H. & Price, T. M. (1999) Presence of leptin in breast cell lines and breast tumors. Biochem. Biophys. Res. Commun. 269:695-698.
38. Smith-Kirwin, S. M., O’Connor, D. M., Johnston, J., Lancey, E. D., Hassink, S. G. & Funanage, V. L. (1998) Leptin expression in human mammary epithelial cells and breast milk. J. Clin. Endocrinol. Metab. 83:1810-1813.
39. Hu, X., Juneja, S. C., Maihle, N. J. & Cleary, M. P. (2002) Leptin—a growth factor in normal and malignant breast cells and for normal mammary gland development. J. Natl. Cancer Inst. 94:1704-1711.
40. Rayner, D. & Trayhurn, P. (2001) Regulation of leptin production: sympathetic nervous system interactions. J. Mol. Med. 79:8-20.[Medline]
41. Lu, Y. P., Lou, Y. R., Lin, Y., Shih, W. J., Huang, M. T., Yang, C. S. & Conney, A. H. (2001) Inhibitory effects of orally administered green tea, black tea, and caffeine on skin carcinogenesis in mice previously treated with ultraviolet B light (high risk mice): relationship to decreased tissue fat. Cancer Res 61:5002-5009.
42. Spady, T. J., Harvell, D. M., Lemus-Wilson, A., Strecker, T. E., Pennington, K. L., Vander Woude, E. A., Birt, D. F., McComb, R. D. & Shull, J. D. (1999) Modulation of estrogen action in the rat pituitary and mammary glands by dietary energy restriction. J. Nutr. 129:587S-590S.
43. Vanden Heuvel, J. P. (1999) Peroxisome proliferator-activated receptors: a critical link among fatty acids, gene expression and carcinogenesis. J. Nutr. 129:575S-580S.
44. Okuno, A., Tamemoto, H., Tobe, K., Ueki, K., Mori, Y., Iwamoto, K., Umesono, K., Akanuma, Y., Fujiwara, T., Horikoshi, H., Yazaki, Y. & Kadowaki, T. (1998) Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. J. Clin. Investig. 101:1354-1361.[Medline]
45. Lin, Y., Kreeft, A., Schuurbiers, J.A.E. & Draijer, R. (2000) Different effects of conjugated linoleic acid isomers on lipoprotein lipase activity in 3T3–L1 adipocytes. J. Nutr. Biochem. 12:183-189.
46. Brown, M., Evans, M. & McIntosh, M. (2001) Linoleic acid partially restores the triglyceride content of conjugated linoleic acid-treated cultures of 3T3–L1 preadipocytes. J. Nutr. Biochem. 12:381-387.[Medline]
47. Brown, J. M., Halvorsen, Y. D., Lea-Currie, Y. R., Geigerman, C. & McIntosh, M. (2001) Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue. J. Nutr. 131:2316-2321.
48. 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.
49. Evans, M., Geigerman, C., Cook, J., Curtis, L., Kuebler, B. & McIntosh, M. (2000) Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3–L1 preadipocytes. Lipids 35:899-910.[Medline]
50. West, D. B., Blohm, F. Y., Truett, A. A. & Delany, J. P. (2000) Conjugated linoleic acid persistently increases total energy expenditure in AKR/J mice without increasing uncoupling protein gene expression. J. Nutr. 130:2471-2477.
51. Park, Y., Storkson, J. M., Albright, K. J., Liu, W. & Pariza, M. W. (1999) Evidence that the trans-10, cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids 34:235-241.[Medline]
52. Satory, D. L. & Smith, S. B. (1999) Conjugated linoleic acid inhibits proliferation but stimulates lipid filling of murine 3T3–L1 preadipocytes. J. Nutr. 129:92-97.
53. Ip, M. M., Zangani, D., Masso-Welch, P. A., Ip, C., Shoemaker, S., Liu, D. M. & Lisafeld, B. (2001) Conjugated linoleic acid acts directly on the mammary epithelium and indirectly through the mammary stroma to prevent breast cancer. J. Nutr. 131:1895-1895.
54. Evans, M., Park, Y., Pariza, M., Curtis, L., Kuebler, B. & McIntosh, M. (2001) Trans-10, cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor 2 and aP2 expression in 3T3–L1 preadipocytes. Lipids 36:1223-1232.[Medline]
55. Ohnuki, K., Haramizu, S., Oki, K., Ishihara, K. & Fushiki, T. (2001) A single oral administration of conjugated linoleic acid enhanced energy metabolism in mice. Lipids 36:583-587.[Medline]
56. Delany, J. P. & West, D. B. (2000) Changes in body composition with conjugated linoleic acid. J. Am. Coll. Nutr. 19:487S-493S.
57. Brodie, A. E., Manning, V. A., Ferguson, K. R., Jewell, D. E. & Hu, C. Y. (1999) Conjugated linoleic acid inhibits differentiation of pre- and post-confluent 3T3–L1 preadipocytes but inhibits cell proliferation only in preconfluent cells. J. Nutr. 129:602-606.
58. Rupnick, M. A., Panigrahy, D., Zhang, C. Y., Dallabrida, S. M., Lowell, B. B., Langer, R. & Folkman, M. J. (2002) Adipose tissue mass can be regulated through the vasculature. Proc. Natl. Acad. Sci. U.S.A. 99:10730-10735.
59. Masters, N., McGuire, M. A., Beerman, K. A., Dasgupta, N. & McGuire, M. K. (2002) Maternal supplementation with CLA decreases milk fat in humans. Lipids 37:133-138.[Medline]
60. Cousin, B., Morroni, M., Raimbault, S., Ricquier, D., Penicaud, L. & Casteilla, L. (1992) Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization. J. Cell. Sci. 103:931-942.
61. Ip, C., Singh, M., Thompson, H. J. & Scimeca, J. A. (1994) Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Res 54:1212-1215.
62. Aro, A., Mannisto, S., Salminen, I., Ovaskainen, M. L., Kataja, V. & Uusitupa, M. (2000) Inverse association between dietary and serum conjugated linoleic acid and risk of breast cancer in postmenopausal women. Nutr. Cancer 38:151-157.[Medline]
63. Voorrips, L. E., Brants, H.A.M., Kardinaal, A.F.M., Hiddink, G. J., Van den Brandt, P. A. & Goldbohm, R. A. (2002) Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands cohort study on diet and cancer. Am. J. Clin. Nutr. 76:873-882.
64. 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.
65. Corl, B. A., Barbano, D. M., Bauman, D. E. & Ip, C. (2003) Cis-9, trans-11 CLA derived endogenously from trans-11 8:1 reduces cancer risk in rats. J. Nutr. 133:2893-2900.
66. Brown, J. M., Boysen, M. S., Jensen, S. S., Morrison, R. F., Storkson, J., Lea-Currie, R., Pariza, M., Mandrup, S. & McIntosh, M. K. (2003) Isomer-specific regulation of metabolism and PPAR signaling by CLA in human preadipocytes. J. Lipid Res. 44:1287-1300.
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