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Department of Cell Biology and Human Anatomy, University of California, School of Medicine, Davis, CA
3 To whom correspondence should be addressed. E-mail: nehubbard{at}ucdavis.edu.
ABSTRACT
Animal studies consistently show that dietary conjugated linoleic acid (CLA) reduces mammary tumorigenesis including metastasis. Relatively low concentrations of CLA are required for those effects, and a threshold level exists above which there is no added reduction. We reasoned that the concentration of CLA required to effectively alter mammary tumor metastasis may be dependent on the type of dietary fat because select fatty acids can enhance or suppress normal or malignant cell growth and metastasis. For this study, the diets (a total of 12 different groups) differed in fatty acid composition but not in energy from fat (40%). In experiments involving spontaneous metastasis, mice were fed for 11 wk; in experiments in which mice were injected i.v. with tumor cells, they were fed for 7 wk. Mice were then assessed for the effect of CLA concentration on mammary tumorigenesis. Mammary tumor growth was not altered, but metastasis was significantly decreased when beef tallow (BT) replaced half of a defined vegetable fat blend (VFB). That blend reflects the typical fat content of a Western diet. In addition, that same VFB:BT diet lowered the concentration of CLA required to significantly decrease mammary tumor metastasis from 0.1% of the diet to 0.05%. A diet in which corn oil replaced half of the VFB did not lower the threshold from 0.1 to 0.05%. In vitro, the main fatty acid in vegetable oil, linoleic acid, reduced the efficacy of CLA toxicity on mammary tumor cells in culture. Alternatively, fatty acids normally found in BT, such as oleic, stearic, and palmitic acids, either did not change or enhanced the cytolytic effects of CLA isomers on mouse mammary tumor cells in culture. These data provide evidence that dietary BT, itself with negligible levels of CLA, may increase the efficacy of dietary CLA in reducing mammary tumorigenesis.
KEY WORDS: • conjugated linoleic acid • mouse mammary tumor metastasis • beef tallow
Conjugated linoleic acid (CLA)4 is a group of dienoic derivatives of linoleic acid (LA) that can be found in natural food sources such as milk fat and the meat of ruminant animals. It can also be synthesized in a laboratory. Several forms of CLA are commercially available as supplements and have been consumed with mainly unsubstantiated human health benefits. Although CLA can be described as a functional food in rodents due to its reported anticarcinogenic, antiatherogenic and antidiabetogenic properties, a recent meta-analysis of human studies with CLA showed a marked variation between reports of health-related outcomes (1). The consensus was that CLA did not affect human body weight or body composition, that it could have detrimental effects in terms of altered blood lipid composition and impaired insulin sensitivity, and that it had only limited effects on immune functions (1). Conclusions from that meta-analysis were from studies that assessed CLA primarily on the basis of dietary supplementation with synthetic isomers and did not involve increasing consumption of CLA-rich foods such as milk and ruminant meats. It is unknown whether a natural approach to increasing CLA intake may alter the observed effects in humans or whether other dietary components can alter the efficacy of CLA. Nevertheless, no dietary studies with CLA and human carcinogenesis have been reported, but support for CLA as an anticarcinogenic agent against human cancer cell lines exists in the literature (2–4). Those observations along with the animal studies showing major health benefits of CLA make it important to assess alterations in potency of CLA because dosages in humans may be critical.
Past studies with CLA in animal models yielded critical data with respect to its undeniable efficacy as an antitumorigenic food component. Those studies showed that CLA can effectively reduce a number of steps in rodent mammary tumorigenesis from initiation to metastasis (5–10). However, a CLA threshold level of 1% of the diet above which there is no longer any effect was established (9–11). That threshold level may be due to a limiting step in the capacity to metabolize CLA to some active products that are essential for cancer prevention (11). A minimum level of dietary CLA required to alter tumorigenesis has not been determined because other dietary components may affect its potency. In a previous study, the efficacy of 1% CLA on tumorigenesis in the rat dimethylbenz[a]anthracene model did not appear to be affected by the amount or type of dietary fat (11). In that study, dietary fat composition for the experiments was based on the composition of the typical American diet. We reasoned that the potency of CLA may be altered by the type of dietary fat and that lower levels could be as efficacious as higher levels provided the diet contained less vegetable fat and more animal fat. That reasoning was based on previous studies showing LA, a vegetable fat, as a major component of a tumorigenic diet and oleic acid (OA), a major component of beef tallow (BT), modifying the LA effect (12,13). In addition, preliminary in vitro data indicated that specific fatty acids could alter the mammary tumor cell cytotoxicity of CLA isomers.
MATERIALS AND METHODS
Reagents. Linoleic acid, cis9,trans11- (c9,t11)-, and trans10,cis12- (t10,c12)-CLA for use in vitro were purchased from Cayman Chemical. DMEM, MEM, F-10 media, insulin, transferrin, L-glutamine, vitamin mixture, nonessential amino acid, and sodium pyruvate solution were purchased from GIBCO; fatty acid–free bovine serum albumin, hydrocortisone, and MTT [3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide] were from Sigma Chemical.
Animals and diets. Female BALB/cANN mice, 5 wk old (Charles River Laboratories) previously fed a standard rodent diet were divided into separate dietary groups such that the mean weight per group was approximately the same. Each group was fed their specific diet (described below) for the duration of the experiment. Compositions of the experimental diets are provided (Table 1). They contained 40% of energy as fat, and contained at least the minimum level of recommended nutrients with a constant amount of protein (casein), salts, vitamins, and fiber per kJ (14). The fat sources were a vegetable fat blend (VFB) previously described by other investigators (11), BT, and corn oil (CO). The vegetable oils that were used to prepare the VFB were coconut, cottonseed, soybean, high-oleic sunflower, and palm as well as cocoa butter. The fatty acid composition of the fats used in the diets is described (Table 2). CLA added to some diets was a mixture predominantly made up of c9, t11-CLA (32.5 g/100 g) and t10, c12-CLA (33.5 g/100 g) isomers (provided by Loeders-Crokklan). There were no differences (P > 0.05) in weight gain among the dietary groups in any of the experiments conducted (data not shown).
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Spontaneous metastasis.
The effect of diet on metastasis from primary tumors transplanted into the mammary fat pad, spontaneous metastasis, was assessed as described previously (9). Tumor cells were injected into the mammary fat pads of mice that had been consuming the experimental diets for 4 wk. Latency was assessed as the time between injection and minimally identifiable tumors. While mice continued to consume the experimental diets, tumor growth was monitored twice weekly using a caliper. When the primary tumor of each mouse reached a mean volume of 
500 mm3, mice were anesthetized, and the tumors removed surgically. The surgical procedures were performed under guidelines approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Mice continued consuming the same diets; they were killed 3 wk after surgery and their tissues examined for metastasis. Metastatic tumor burden was calculated by multiplying the number of lung nodules by the mean volume/nodule. There were some mice with extrapulmonary nodules, but no differences were observed among the groups.
Experimental metastasis. To examine the effects of dietary fat on tumor cell lodgment, invasion, and proliferation to form metastases (experimental metastasis), mice fed the experimental diets for 4 wk were then injected i.v. in the tail vein with 1 x 105 line 4526 tumor cells. Mice were injected randomly and all within 1 h; thus, metastasis for all mice had similar initiation times. Mice were returned to their cages and continued to consume the experimental diets for an additional 3 weeks. Mice were then killed and their tissues examined for metastases. Lungs were prepared for assessing metastasis as in the spontaneous metastasis experiments. There were some mice with extrapulmonary nodules, but no differences were observed among the groups.
Protocol. For the first objective, mice in 3 separate groups were fed either a diet that contained VFB as its sole source of fat, a diet containing VFB and BT (1:1) as the fat source, or a diet containing VFB:CO (1:1). Mice in 6 separate dietary groups were fed the diets described in Table 1 for the second objective, and lung tumor burden was evaluated after both spontaneous and experimental metastasis. For objective 3, mice in 6 separate groups were fed diets similar to those described in Table 1 except that the concentration of CLA was 0.05%. Lung tumor burden was evaluated after experimental metastasis. For the last objective, mice in 6 separate groups were fed diets similar to those described in Table 1 except that the ratio of VFB to BT or CO was 3:1. Lung tumor burden was then evaluated after experimental metastasis.
In vitro studies. Viability, proliferation, and apoptosis assays were performed as previously described (17). Cells were grown in MEM, L-glutamine, vitamin mixture, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 200 g/L gentamicin with 5% bovine calf serum at 37°C. Cells were washed with PBS and placed in a defined medium containing DMEM/F-10, 0.01 g/L hydrocortisone, 0.08 g/L insulin, 0.1 g/L transferrin, 10 g/L fatty acid–free bovine serum albumin, and 0.02 g/L gentamicin (18). Fatty acids were combined with ethanol vehicle, which was used as the control. Cell viability was assessed using the MTT assay as previously described (19) and confirmed with trypan blue exclusion. Briefly, wells were seeded with 5 x 104 cells in 24-well plates in media with 5% fetal bovine serum and incubated for 16 h. Cells were then washed with PBS and treated with the fatty acids; the media were then removed and the monolayers incubated with 1 g/L MTT solution in HBSS. The resultant formazan crystals were dissolved in 2-isopropanol and the wells read at an optical density of 550 nm using a plate reader.
Analysis of apoptosis. A commercially available assay was used for this analysis (Cell Death Detection ELISA, Roche Molecular Biochemicals). Briefly, cells were incubated in 24-well plates overnight and treated with fatty acids for 24 h. Cells were incubated with the kit lysis buffer and the cytoplasmic fraction isolated by centrifugation (20,000 x g for 10 min). Microplates were coated with anti-histone antibody, washed, and samples added in triplicate for 90 min then analyzed according to manufacture's instructions.
Cell proliferation assay. Cell proliferation was measured with 5-bromo-2'-deoxyuridine (BrdU) incorporation and the Cell Proliferation ELISA kit (Roche). Briefly, tumor cells were incubated with fatty acids for 8 h and then BrdU for 2 h. Cells were fixed and the DNA denatured for 30 min at room temperature. Samples were incubated with anti-BrdU-peroxidase for 90 min, washed, the kit substrate added, and optical density measured.
Invasion assay.
The effect of fatty acids on the invasion capacity of line 4526 mouse mammary tumor cells was assessed using the QCM 96-well Cell Invasion Assay (Chemicon). In that assay, cultured cells were harvested and added to the invasion chamber of the microplate for 24 h after which the degree of invasion was assessed colorimetrically. Under control conditions, 20% of line 4526 mammary tumor cells invaded the matrix.
Statistical analysis. All data were initially evaluated using 1-way ANOVA. When significant differences were detected, Fisher's Protected Least Significant Difference and the Bonferroni/Dunn procedure were used to assess differences between the groups or treatments (20) with a prespecified P-value of 0.05. Because metastasis data are usually not normally distributed, we also used the Kruskal-Wallis nonparametric test for its analysis. Nevertheless, results obtained were similar when ANOVA was used for that data set.
RESULTS
Dietary fat and CLA reduction of metastasis. Dietary fat and CLA did not affect latency or primary tumor growth rate (data not shown). That lack of effect of CLA on primary tumor growth rate but not metastasis could be due to an effect on one or more steps in the metastatic cascade such as dissemination, lodgment, extravasation, or proliferation. In our first objective, the effect of dietary fat composition on lung tumor burden was assessed. Mice fed the diet containing less VFB due to replacement with BT had a lower (P < 0.05) burden than mice fed the diet containing only VFB as the fat source (Table 3). When CO replaced half of the fat, the lung tumor burden was not altered compared with VFB only but was increased (P < 0.05) compared with VFB:BT. Because the diets contained the same amount of fat by weight (20%) and energy (40%), the effects were likely due to fat composition. In our second objective, CLA added at 0.1% did not alter pulmonary tumor burden in mice that had spontaneous metastases from primary tumors (data not shown). However, when experimental metastasis was assessed, 0.1% CLA decreased (P < 0.05) lung tumor burden in both groups fed the VFB only or VFB:BT diets, but not in those fed the VFB:CO diet (Fig. 1). Therefore, CLA was more effective at decreasing the lung tumor burden when BT rather than CO was present in the diet.
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DISCUSSION
Conjugated linoleic acid is a functional food, at least in rodents, due to its ability to potently inhibit tumorigenesis. Although humans consume CLA in various quantities either naturally or as a synthetic supplement, there is no conclusive evidence that it is beneficial. However, none of the human studies controlled for the composition of dietary fat nor were conclusions made about how dietary fat composition could alter CLA efficacy. In this study, we assessed how the potency of CLA in decreasing tumor metastasis, may depend on dietary fat composition. The effect of dietary fat composition on CLA efficacy was tested previously but was focused on mammary tumor incidence in a rat mammary carcinogenesis model (11). The results of that study suggested that the efficacy of CLA was not altered by the amount or type of dietary fat. Our assessment was based on previous studies in which mammary tumor metastasis was significantly altered in a manner dependent upon dietary fat composition or the presence of CLA (9,10). We assessed metastasis because preventing its progression results in a more favorable outcome and because it is that stage of tumor progression that most often kills the host. In this report, we showed that replacing a portion of a vegetable fat blend that imitates the dietary fat composition of a typical American diet with BT not only decreased mammary tumor metastasis but significantly increased the potency of CLA. That conclusion is based on 0.05% CLA decreasing metastasis when only BT was used to replace half of the vegetable fat blend. With 0.1% CLA, there was a decrease in metastasis even with the diet containing only the vegetable fat blend as the source of fat. Those observations were made only after metastasis was assessed when tumor cells were injected into mice i.v. We did not see an effect of 0.1% CLA on metastasis in mice that had first grown primary tumors in any of the dietary groups. This could be due to low levels of CLA having effects only on those steps of the multistage process of metastasis that occur after cells have entered the blood stream, such as lodgment, survival, and proliferation. After replacing half of the VFB with BT, the dietary fat composition was significantly altered. Specifically, the levels of SA, PA and OA were increased and the level of LA was decreased. We previously showed that OA could alter the LA enhancement of mammary tumor metastasis (13). In this study, replacing a portion of VFB with CO did not alter metastasis but actually decreased the potency of 0.1% CLA. We showed previously that increasing the level of LA in a diet with 40% of energy as fat could increase the level of metastasis in a dose-dependent manner in this mammary tumor model (12). In this study, replacing half of the VFB with CO significantly increased the level of LA in the diet and thus may have been responsible for decreasing CLA potency. Even replacing 25% of the VFB with CO diminished the efficacy of 0.1% CLA in reducing mammary tumor metastasis. These in vivo findings suggest that the composition of dietary fat may alter the effectiveness of CLA. That is important because voluntary CLA supplementation in the U.S. diet may be on the increase due to the reported body fat loss in CLA-supplemented animals. Unfortunately, human studies to date are unable to confirm the animal results. There is a possibility that too high a level of CLA supplementation could be detrimental. Therefore, finding a nontoxic and effective level is important.
In addition to the in vivo results, we reported that specific fatty acids may alter in vitro CLA effects. The isomers we used in the feeding studies were the 2 most often used in other studies and the ones most often present in commercial preparations. Although the c9,t11-CLA isomer appears to be the one found in the highest concentrations in natural sources and has potent biological activities, the t10,c12-CLA isomer occurs naturally in much lower concentrations but also has potent biological effects when added in vitro and as an in vivo supplement. We and others showed previously that CLA isomers added to tumor cells in vitro significantly decrease their viability. Those effects were reviewed recently (21). To our knowledge, none of the in vitro studies with CLA isomers have assessed the effects of other fatty acids on the efficacy of CLA in decreasing tumor cell viability. In the transplantable model with mouse mammary tumor cell line 4526, we were able to assess the effects in vitro on the same cells used in the in vivo studies. In our previous study, the t10,c12-CLA isomer alone decreased the viability of line 4526 mammary tumor cells by as much as 60% (17). That decreased viability appeared to be due to a combination of increased apoptosis and decreased tumor cell proliferation. In this study, we incubated tumor cells with combinations of CLA isomers and fatty acids found in the fats used in the dietary studies. Specifically, we assessed OA, PA, and SA, which make up a large portion of the fatty acid composition of BT, and LA, which makes up, for example, 60% of CO. Interestingly, we found that palmitic acid decreased tumor cell viability, albeit to a lesser extent than t10,c12-CLA. Palmitate was found previously to stimulate apoptosis in caspase 3–deficient MCF-7 human mammary tumor cells (22). That effect was also seen in a normal insulin-secreting cell line (23). When those 2 fatty acids were combined, tumor cell viability was decreased in an additive fashion and was not synergistic. Those observations are consistent with observations in vivo. Beef tallow is made up of 25% PA; thus, replacing half of the VFB with BT results in quite a large increase in the PA content and may be important in boosting CLA potency. Surprisingly, the addition of OA in conjunction with CLA isomers in vitro did not alter tumor cell viability. BT is made up of 50% OA; thus, the OA content of the VFB:BT blend would be significantly increased. Perhaps the effect of OA in vivo is related to its possible effects on the "soil" or tissues to which tumor cells metastasize rather than the "seed" or tumor cells.
Alternatively, we showed that LA partially inhibited the ability of t10,c12-CLA to decrease tumor cell viability. That is also consistent with our in vivo observations in this report. Corn oil is made up of 60% LA; replacing half of the VFB, which already contains a large quantity of LA, with CO, boosts the LA content to a relatively high level.
In conclusion, we reasoned that the effects of CLA could be altered by dietary fatty acid composition because several fatty acids had quite diverse biological effects. In vivo, CLA seems to be more effective with respect to decreasing metastasis when the diet includes beef tallow. On the other hand, the efficacy of CLA is diminished with increased levels of dietary LA found in several popular vegetable oils. Although metastasis is a complicated process that involves more than simply the survival of the tumor cell, direct effects on tumor cells can alter survival and invasive capacity. These results are important when CLA is considered for antitumor therapies because its intake as a dietary supplement is increasing steadily.
ACKNOWLEDGMENTS
We thank Dr. Peter Friedman with ACH Foods (Memphis, TN) for providing cocoa butter, beef tallow, soybean, palm, high-oleic sunflower, cottonseed, and coconut oils.
FOOTNOTES
1 Published in part in abstract form [Hubbard NE, Lim D, Erickson KL. Antitumor effects of conjugated linoleic acid may depend on fatty acid composition (abstract). Proc Am Assoc Cancer Res. 2005;46:3469]. 
2 Funded by beef and veal producers and importers through their $1-per-head checkoff and was produced for the Cattlemen's beef board and state beef councils by the National Cattlemen's Beef Association.
4 Abbreviations used: BT, beef tallow; BrdU, 5-bromo-2'-deoxyuridine; CLA, conjugated linoleic acid; CO, corn oil; LA, linoleic acid [18:2(n-6)]; MTT [3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide]; OA, oleic acid [18:1(n-9)]; PA, palmitic acid (16:0); SA, stearic acid (18:0); VFB, vegetable fat blend.
Manuscript received 5 August 2005. Initial review completed 23 September 2005. Revision accepted 23 October 2005.
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