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© 2007 American Society for Nutrition J. Nutr. 137:1423-1429, June 2007

Nutrition and Disease

Conjugated Linoleic Acid Alters Matrix Metalloproteinases of Metastatic Mouse Mammary Tumor Cells1,2

Neil E. Hubbard*, Debora Lim and Kent L. Erickson

Department of Cell Biology and Human Anatomy, University of California School of Medicine, Davis, CA 95616-8643

* To whom correspondence should be addressed. E-mail: nehubbard{at}ucdavis.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Conjugated linoleic acid (CLA) is a group of linoleic acid derivatives that has been implicated in animal studies to reduce a number of components of mammary tumorigenesis. Previously, we showed that CLA could alter the latency and metastasis of the highly metastatic transplantable line 4526 mouse mammary tumor. Several possible mechanisms have been proposed for the actions of CLA, but here we assessed how CLA may act to alter the expression and activity of matrix-modifying proteins within tumors from line 4526. In vitro, highly metastatic mouse mammary tumor cells had significantly decreased invasiveness after treatment with CLA, an indication that matrix-modifying proteins may have been altered. Using these same highly metastatic cells, primary tumors were grown in mice of separate groups fed 0, 0.1, 0.5, and 1% CLA (wt:wt) and evaluated for their levels and activities of matrix-modifying enzymes, enzyme inhibitors, and enzyme activators. The addition of CLA to the diet increased steady-state levels of messenger RNA (mRNA) of the matrix metalloproteinases (MMP) -2 and -9 in primary tumors removed from mice. However, western analysis revealed that although relative levels of the proform of MMP-9 were consistent with the mRNA observations, MMP-2 proform levels were actually decreased by dietary CLA. The activity of MMP-2 was barely detectable, but gelatin zymography and an in vitro activity assay showed that MMP-9 activity was significantly decreased by CLA. The steady-state mRNA and protein levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2, natural inhibitors of MMP, were increased at higher dietary CLA levels relative to low or no CLA. Suppression of MMP activity, therefore, may be 1 pathway through which CLA reduces tumor invasion and spread.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Evidence from many animal studies now supports the idea of a protective role of dietary conjugated linoleic acid (CLA)3 intake in mammary tumorigenesis (1,2). CLA refers to a group of isomers of linoleic acid (LA). Two such isomers that have been the focus in many of the studies are 9c, 11t-18:2 and 10t, 12c-18:2 (3). Similar effects of decreased tumorigenesis have been shown with dietary vaccenic acid, which is converted in animal tissues to CLA (4). Other CLA precursors have been found in vegetable sources such as tung oil ({alpha}eleosteric acid), perilla oil ({alpha}linolenic acid), and pomegranate seed oil (punicic acid), but those fatty acids need further testing for efficacy against mammary tumorigenesis (5). Therefore, the potential for altering dietary intake to change CLA tissue status as a means for decreasing risk of mammary tumorigenesis is a distinct possibility.

Whereas CLA fed to experimental animals has been shown to have a wide range of effects, such as reduction of body fat, inhibition of atherosclerosis, and alteration of tumorigenesis (6,7), a number of the CLA effects has been shown in rodent models of tumorigenesis. For example, as little as 0.1% CLA in the diet reduced the appearance of mammary tumors after administration of a carcinogen (8). The response to CLA was maximal at the relatively low concentration of 1%, which may have important implications, because that amount could theoretically be incorporated into the human diet. Dietary CLA had a protective effect against tumor initiation when fed to mice from time of weaning until carcinogen exposure (8). This CLA treatment corresponds to the time of mammary gland maturation. When CLA feeding began after exposure, an inhibition of tumorigenesis was also observed (8). Thus, CLA had a direct chemoprotective effect on the mammary gland during development as well as a suppressive influence on tumor progression. Our previous work showed that mixed CLA isomer intake affected the latency, metastasis, and secondary tumor burden in mice with highly metastatic line 4526 mammary tumors (9). Although the tumor growth rate was not changed, onset of tumor initiation was delayed by 10–15 d. More importantly, the number and size of nodules that metastasized to the lung was significantly decreased. This suggests that CLA may also affect later stages of tumorigenesis, especially metastasis (9). In addition, we showed that the total composition of dietary fat could alter the effectiveness of CLA in reducing breast tumor metastasis (10).

The mechanisms through which CLA exerts its effects are largely unclear. Numerous studies have indicated that metastasizing tumor cells need to express 1 or several proteinases to aid in the destruction and remodeling of surrounding tissues and migration to a secondary site. Several types of proteinases play an important role in the remodeling of the extracellular matrix (ECM) in a variety of physiological and pathological conditions. These include serine proteinases, cysteine proteinases, and the matrix metalloproteinases (MMP). MMP are a family of zinc endopeptidases that are secreted as proenzymes and have proteolytic activity toward components of the ECM. Some MMP, such as gelatinases and membrane type (MT) 1 MMP, are localized to leading edges during normal and neoplastic cell invasion and migration. In particular, MMP-2 and MMP-9, members of the gelatinase subfamily of MMP, are believed to play a critical role in tumor invasiveness and metastasis. These MMP are able to degrade collagen type IV, a major component of basement membranes, and are found at many sites where tissue remodeling occurs. They can be produced by reactive stromal cells around breast tumors and by several breast cancer cell lines (11).

The expression of these proteinases is tightly regulated and subject to several layers of control in transcription and posttranslational activation (12). In addition, their activity can be inhibited by other proteins, generally through tissue inhibitors of metalloproteinases (TIMP) (13). The role of MMP, TIMP, and MT-MMP in cancer metastasis has recently been reviewed (14). Most MMP are secreted in a latent form and can be activated through cleavage by other MMP and by serine protease, such as trypsin or plasmin (15,16). MMP-9 can also be activated by other MMP, such as MMP-2 and MMP-3 (17,18). However, MMP-2 is unique in that it is activated by membrane-bound MMP (19). The proform of MMP-2 activation by MT1-MMP (also known as MMP-14) is dependent on small quantities of TIMP-2, which forms a ternary complex that is bound to the membrane. Another MT1-MMP near the complex then cleaves the bound MMP-2. At high concentrations of TIMP-2, MMP-2 is not activated, because the inhibitor blocks MT1-MMP activity. Other MT-MMP can also independently activate MMP-2, such as MT2-MMP (MMP-15).

Recent work has shown the existence of a correlation between MMP-2 and MMP-9 expression and the metastasis and invasion of several types of human carcinoma (20). In this study, we sought to determine how dietary CLA alters tumor cell metastasis by examining modulation of MMP-2 and MMP-9 expression. That may occur by alteration of the endogenous inhibitors, TIMP-1 and TIMP-2. This may lead to reduced invasiveness and thus metastasis. We have already demonstrated in mice that CLA can alter tumor metastasis, a process that involves invasion. Though we do see an alteration of tumor cell invasiveness in vitro by CLA, we chose to focus our MMP studies on tumors grown in mice fed various concentrations of CLA.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Mice and diets. We obtained 5-wk-old female BALB/cANN mice from Charles River and randomly segregated into groups for the diet studies. After 48 h, mice fed a standard stock diet (Purina) were switched to 1 of the 4 experimental diets (Table 1) (21). All diets contained 20% (wt:wt) total fat, mostly from corn oil, and differed only in the amount of CLA, as described previously (9). The CLA was a generous gift from Loders and Croklaan and was added at concentrations of 0.1, 0.5, or 1.0% (wt:wt) in place of corn oil. This CLA was a mixture of isomers, with predominantly 9c, 11t-18:2 (32.5 g/100 g) and 10t, 12c-18:2 (33.5 g/100 g). The remainder was other isomers (16 g/100 g) and 18:1 (18 g/100 g). Mice were fed fresh diet daily for the duration of the study. All diets were isoenergetic and contained at least the minimum level of recommended nutrients with a constant amount per kilojoule of casein, salts, vitamins, and fiber (22). The contents of the vitamin mix have been published previously (21). We weighed mice weekly to monitor growth; dietary groups did not differ (P < 0.05). Mice were handled in accordance with the standards set forth by the University of California Davis Animal Use and Care Administrative Advisory Committee under strict guidelines set forth by the Association for the Assessment and Accreditation of Laboratory Animal Care, International.


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  		TABLE 1 Composition of semipurified experimental diets with or without CLA

 
    Tumor cells and transplantation. The highly metastatic mouse mammary tumor cell line 4526 is a cloned subpopulation derived from a spontaneously arising mouse mammary adenocarcinoma of a BALB/cfC3H mouse (23). Tumor cells were maintained in culture as previously described (10). After 3 wk of feeding the experimental diets, 5 x 104 tumor cells were prepared as previously described (10) and transplanted subcutaneously without surgery into 1 mammary fat pad of each mouse. Mice were prepared for injection by shaving the hair over the fat pad and increasing the transparency of the skin with 70% ethanol. With that procedure, tumors grow within the fat pad 99% of the time. Mice were checked daily after transplantation until tumors appeared (latency) and then monitored for tumor growth twice weekly. Tumors that grew outside of the fat pad or invaded the peritoneum were not used in this study. In previous studies over a span of 20 y with tumors that arise from transplantation of these cells, we have observed little variability with respect to latency and growth (10,24,25). When the primary tumors reached a mean volume of ~500 mm3, they were removed and snap-frozen in liquid nitrogen and stored at –80°C. With that volume of primary tumor, cells begin to metastasize.

    Cell invasion assay. The highly metastatic mouse mammary tumor cell line 4526 was assayed for invasive potential using the QCM 96-well Cell Invasion assay kit (Chemicon). Tumor cells in culture were treated for 24 h in serum-free conditions with fatty acids. Ethanol was used as a vehicle control (0.05%). A duplicate culture dish was used to assess viability after treatment (26). Tumor cells for the assay were harvested and resuspended in media without chemoattractant and with fatty acids. After adjusting for viability, 1 x 104 viable cells were added to each invasion chamber. Media containing 10% fetal bovine serum was used as a chemoattractant in the assay. Cells that migrated through the ECM layer after 24 h were disassociated from the bottom membrane by incubation with the cell detachment buffer provided with the kit. The migrated cells were then lysed and quantitated by fluorescence with a nucleic acid dye, CyQuant GR (Invitrogen). To estimate the number of cells that had migrated in the samples, a standard curve was prepared using lysates of known cell quantities.

    Preparation of RNA and RT-PCR. Total RNA was isolated from tumor lysate with the Qiagen RNeasy kit (Qiagen) according to the manufacturer's instructions. The concentration of total RNA was measured using RiboGreen RNA Quantitation (Invitrogen). cDNA was generated by RT using an oligo(dT) primer and the Superscript First-Strand Synthesis system (Invitrogen). Real-time semiquantitative PCR was performed using a LightCycler (Roche Molecular Biochemicals) as described previously (27). The oligonucleotide primer sequences were synthesized based on previously published reports for MMP-2, MMP-9, and TIMP-1 (28); MT1-MMP (29); MT2-MMP (30); TIMP-2 (31); ßactin (32); and glyceraldehyde-3-phosphate dehydrogenase (33). An aliquot of the final product was run on a 2% agarose gel for further verification of amplification and size. The LightCycler analysis program used the curves generated from fluorescence monitoring to calculate a crossing point (CP), which was used for sample comparison. Relative copy number (RCN) was determined by the formula 2[CP(experimental)– CP(housekeeping)]. Because all our experiments described results relative to control values, we assumed an efficiency of 100% and thus a doubling every cycle.

    Western analysis. Western analysis was performed essentially as described (27). Briefly, tumor tissue lysates were prepared by homogenization in cold lysis buffer, centrifuged to remove particulate matter, and the protein concentration of the resulting supernatant determined using the Bio-Rad Protein Assay. Samples were electrophoresed under denaturing conditions in a 12% Novex Tris-glycine gel. After transfer to polyvinylidene fluoride membranes (Pall Life Sciences), incubation with antibodies (Santa Cruz Biotechnology) and amplification and detection using a biotinylated secondary antibody and phosphatase-labeled streptavidin, bands were visualized after development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride (Sigma). Lysates from separate tumors of each dietary group were used to make 3–5 different westerns dependant upon the protein assessed. Bands of westerns were quantitated by area and density using Scion Image.

    Gelatin zymography. We prepared protein lysates as described above for western analysis. Standards and samples were mixed with an equal volume of sample buffer and electrophoresed on a 10% Novex gelatin zymogram gel (Invitrogen). Gels were then incubated with renaturing buffer (Invitrogen) for 30 min at room temperature, then overnight at 37°C with developing buffer (Invitrogen). Gels were then stained with 0.5% Coomassie and destained with 5% acetic acid and 25% isopropanol. Areas of gelatinase activity were visible as clear bands on a dark blue background. Bands on zymograms were quantitated by area and density using Scion Image.

    MMP activity assay. Tumor samples were also assayed for MMP activity using the Biotrak Matrix Metalloproteinase Activity Assay system (Amersham Pharmacia) and prepared as recommended by the manufacturer. Briefly, tumor tissue was homogenized in cold 50 mmol/L Tris-HCl buffer, pH 7.4, with 1 mmol/L monothioglycerol, passed through a 20-gauge needle, then centrifuged at 2000 x g; 10 min. The cleared lysate was measured for total protein concentration before use in the assay. The samples were tested for actual MMP activity and latent (total) MMP activity after treatment with the synthetic activator p-aminophenyl mercuric acetate (APMA). The assay used a detection enzyme that was activated by captured active MMP, which then cleaved to a chromogenic peptide substrate.

    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 (34) with a prespecified P-value of 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Effect of CLA on invasive potential of mammary tumor cells. We showed previously that mice fed diets containing CLA exhibited a reduction in the number and size of pulmonary metastases (9,25). In addition, CLA reduced the lodgment, survival, and proliferation of mammary tumor cell metastasis (9,25). To begin assessment, we tested in an invasion chamber the effect of varying concentrations of fatty acids on the migration of tumor cells through an ECM. At a concentration of 10 nmol/L, CLA decreased (P < 0.05) the number of invasive cells compared with control and to cells treated with 10 nmol/L LA, arachidonic acid (AA), or oleic acid (OA) (Fig. 1). At higher concentrations (100 nmol/L and 1000 nmol/L) of CLA, the invasive potential of treated cells was decreased even further, as the other fatty acids either had no further effect or increased invasion capacity. That CLA effect was not due to toxicity, because only viable tumor cells treated with the fatty acids were used in the assay. This suggests that the reduced in vivo metastasis observed with dietary CLA may be due to an altered tumor cell invasion and migration capacity.


Figure 1
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  		FIGURE 1  The effects of various fatty acids on mouse mammary tumor cell invasion in vitro. Invasiveness was measured over a 24-h time period. The control value represents cells not incubated with fatty acids. Values are means ± SEM, n = 3. Means for a concentration without a common letter differ, P < 0.05.

 
    Effect of CLA on transcripts of proteases, activators, and anti-protease. Because invasion and migration capacity of tumor cells is partly dependent on expression of ECM-modifying enzymes, we assessed the changes in steady-state messenger RNA (mRNA) levels of the proteases MMP-2 and MMP-9, MMP activators MT1-MMP and MT2-MMP, as well as the antiproteases TIMP-1 and TIMP-2 in tumors removed from mice fed experimental diets. Those genes selected have previously been implicated in tumor progression and metastasis (35). The RCN of MMP-2 transcripts was increased (P < 0.05) in tumors from mice fed the CLA diets compared with transcripts of tumors from mice fed the control diet (Fig. 2A). A similar result was observed with transcripts of MMP-9, which had a nearly 2 times greater level in tumors of mice fed CLA compared with the control. Transcripts for MT1-MMP, an activator of MMP-2, was also increased (P < 0.05) in the tumors of mice fed CLA (Fig. 2B). MT2-MMP, another activator of MMP-2, had decreased (P < 0.05) mRNA levels in tumors of mice fed CLA. When the level of transcripts for the inhibitors of MMP-2 and MMP-9 (TIMP-1 and TIMP-2) were examined, there was a marked increase in tumors of mice fed CLA compared with control mice (Fig. 2C). Transcripts for TIMP-1 were increased between 50 and 100% in tumors of mice fed 1% CLA compared with control mice. Transcripts for TIMP-2 were also increased in tumors of mice from all the CLA groups.


Figure 2
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  		FIGURE 2  Transcript levels of MMP proteases (A), activators (B), and antiproteases (C) in mouse mammary tumors of mice fed diets containing various CLA concentrations. RCN was quantitated as the ratio of the specific mRNA in the experimental diet groups relative to the control group (0% CLA). Values are means ± SEM, n = 5–8. Means without a common letter differ, P < 0.05.

 
    Western analysis of MMP, their activators, and inhibitors. Most MMP are translated as inactive precursors that are then processed to become fully activated. MMP-2 protein was clearly present in lysates of tumors from mice of each dietary group (Fig. 3A). The overall amount of active protein was less than the latent proform in all the diets. The amount of MMP-2 proform decreased (P < 0.05) in tumors of mice fed 1% CLA compared with control diet and 0.1% CLA diets (Fig. 3A,B). TIMP-2 was also detected in tumors of mice from all dietary groups, as was MT1-MMP and MT2-MMP. The protein levels of TIMP-2 were greater (P < 0.05) in tumors of mice fed 0.5 and 1.0% CLA compared with control. Both the latent and active forms of MMP-9 were detected in all tumors. The proform of both MMP-9 and TIMP-1 increased (P < 0.05) in tumors of mice fed 0.5 and 1.0%. Bands representing the active form of MMP-9 were much lighter than the bands representing the proform and were unaltered by CLA. Because CLA altered MMP-9 proform, but not active enzyme, suggests a CLA-induced deficit in MMP-9 activation.


Figure 3
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  		FIGURE 3  Western analysis of mouse mammary tumor proteins from mice fed diets containing various CLA concentrations (A) and relative comparison in the various CLA concentrations for each of the quantitated bands (B). For quantitation, arbitrary units were calculated from the area and density of each band multiplied by 100. Valid comparisons were made in only the different dietary treatments of the same protein. Values are means ± SEM, n = 3–5, dependent on the protein probed. Means for a particular protein without a common letter differ, P < 0.05.

 
    Effect of CLA on tumor gelatinase activity. Because the western analysis indicated that the presence of the protein proform did not coincide with the level of the active form, we further assessed protein expression by measuring gelatinase activity. Four clear bands for each tumor lysate sample were visible by zymography (Fig. 4A), 1 corresponding to the 68-kDa MMP-2 proenzyme (36). A band below that, estimated at 60 kDa, corresponded to the active form of MMP-2 (36). The bands of active MMP-2 were less intense than those of the proenzyme with decreased (P < 0.05) amount of proform at 0.5% (Fig. 4B). Two other bands were visible on the zymogram, with 1 band appearing at ~98 kDa, which corresponds to standards for the proenzyme form of mouse MMP-9 (37) and another appearing at ~88 kDa, which corresponds to the expected size for the activated form of MMP-9 (37). Similar to MMP-2, the intensity of the band representing the activated form of MMP-9 protein was less than that of the proenzyme form. The gelatinase activity of the latent form of the MMP-9 increased (P < 0.05) at the higher dietary levels of CLA; however, the active form decreased (P < 0.05) in response to all CLA treatments (Fig. 4B).


Figure 4
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  		FIGURE 4  Zymographic analysis of gelatinase activity in mammary tumors of mice fed diets containing various concentrations of CLA (A) and analysis and comparison of relative band densities (B). For band quantitation, arbitrary units were calculated from the area and density of each band multiplied by 100. Active gelatinases appear as cleared bands after Coomassie Blue staining. The 0% CLA treatment was the designated control value for each protein. Values are means ± SEM, n = 3. Means for a protein without a common letter differ, P < 0.05.

 
    Effect of CLA on MMP activity. Levels of active MMP-2 were barely detectable with an antibody-based assay system (Fig. 5A) regardless of the concentration of CLA in the diet. When antibody-bound proteins were treated with APMA, MMP-2 was activated and detected by the assay. Therefore, most of the MMP-2 present in the lysate was in an inactive form. The amount of total MMP-2 increased (P < 0.05) in tumors of mice fed 0.5 and 1.0% CLA compared with the control mice (Fig. 5A). The total MMP-9 measured (after APMA treatment) in tumors of mice fed the various concentrations of CLA did not differ (P > 0.05); however, the amount of active MMP-9 decreased (P < 0.05) as dietary CLA increased (Fig. 5B), which implied that CLA could alter the activation of the enzyme.


Figure 5
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  		FIGURE 5  Comparison of active and total MMP-2 (A) and active and total MMP-9 (B) of mouse mammary tumors from mice fed diets containing various concentrations of CLA. Total MMP was measured after treatment with APMA to activate all captured proteins, as described in "Materials and Methods." Values are means ± SEM, n = 3. Means for a protein without a common letter differ, P < 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Previously, we showed that dietary CLA could alter the metastasis of a mouse transplantable mammary tumor in BALB/C mice (9). Most importantly, the number and size of the secondary lung metastases were significantly reduced. Our results here show that CLA may accomplish that by altering the invasive potential of a highly metastatic mouse mammary tumor cell line. In a recent study with human gastrointestinal tumor cells, CLA inhibited invasion into type IV collagen-coated membranes (38). In that same study, direct injections of CLA into the peritoneum inhibited peritoneal metastasis of human tumor cells injected into nude mice.

For us to understand how CLA inhibited pulmonary metastasis, we focused on CLA-altered expression of proteases and antiproteases by the tumors, specifically MMP-2 and MMP-9. These MMP have been shown to alter tumor cell migration, growth, and invasion, and CLA has been shown to have chemoprotective effects. In this study, we showed dietary CLA decreased the activity of MMP-9 in tumors. That may be due to the increased production of MMP inhibitors and decreased activators.

MMP have been implicated as major components in tumor cell growth, migration, and invasion (20,35,39,40). For example, in glioma cells, increased tumor grade was linked to constitutively expressed MMP-2 and MT1-MMP (41) as well as MMP-9 (42). The production of TIMP, however, may be just as important as MMP, because evidence suggests that they are more than just inhibitors (43). TIMP are multifunctional proteins that play a role in the regulation of cell proliferation, apoptosis, and angiogenesis. Those effects may occur through MMP-dependent or -independent pathways. Moreover, TIMP may have both inhibitory and stimulatory effects on tumorigenesis when overexpressed in malignant cells or in mice (44). In several human tumor types, elevated TIMP levels were associated with cancer progression and identified as a marker for poor prognosis (45).

We showed that dietary CLA can alter MMP in primary tumors. Steady-state levels of MMP-2 mRNA and the amount of total MMP-2 protein increased with CLA in the diets. MMP-2 activity in tumor lysates, however, was barely detectable. When transcripts of MMP-2 activators were measured, levels of MT2-MMP decreased although MT1-MMP increased. Because the protein levels of these 2 activators did not appear to be significantly changed by diet, it was likely that the inhibitor, TIMP-2, could play a role in the decreased MMP-2 activity. Transcripts of TIMP-2 were enhanced by the addition of CLA to the diet. We showed that protein levels of TIMP-2 increased only slightly with CLA treatment. However, we did not assess whether the TIMP-2 measured was free or previously bound to MT1-MMP or active MMP-2, even though gels were run under reducing conditions. TIMP-2 bound to MT1-MMP or MMP-2 could explain, though, the discrepancy between the measured transcripts and proteins. This implies that CLA may affect the activation of the MMP-2 proenzyme, perhaps by altering the production of MMP-2 activators or inhibitors, or both.

Most MMP are secreted as inactive precursors and require the proteolytic removal of the N-terminal propeptide to become catalytically active. For MMP-9, this process involves other MMP and serine proteases (46,47), specifically MMP-2 and MMP-3, or MMP-2 in conjunction with MT1-MMP and TIMP-2. MMP-9 transcripts increased with the high dietary concentration of CLA, but diet had an inverse effect on the activity of the protein. In the control group, almost all the detectable MMP-9 protein was active, but the activity of MMP-9 decreased significantly with higher concentrations of CLA, although the amount of total MMP-9 remained consistent. In this case, it may be that CLA exerts its influence not only on the activation of the proenzyme but may additionally influence the translation of MMP-9. Active MMP-9 could also be inhibited by TIMP-1. Levels of TIMP-1 mRNA significantly increased with the higher CLA diets. Protein levels of TIMP-1 also increased. Both of those factors, the increase in inhibitor TIMP-1 and the lack of active MMP-2 as an activator, may explain 1 pathway by which CLA depressed the activity of MMP-9.

All of the specific mechanisms by which CLA inhibits tumorigenesis have not yet been clearly elucidated. Other researchers have proposed that CLA may indirectly exert many of its physiological functions by modulating the accumulation of AA in phospholipids. That could result in a reduced arachidonate pool and reduced production of downstream eicosanoid products. Interestingly, AA, LA, and OA significantly increased tumor cell invasion in our study. Colorectal tumor proliferation has been shown to be stimulated by prostaglandin E2, a derivative of AA (48). In addition, AA potently stimulated malignant epithelial cellular invasion in a recent study using the prostate cancer cell line PC-3 (49). LA can be converted to AA by mammalian tissues and has been shown to significantly increase metastasis (24). Data with OA is a bit more ambiguous. For example, although OA was shown to partially inhibit the formation of lung metastatic colonies, an affect on MMP was not apparent (50). Also, OA can enhance the proliferation of prostate cancer cell line DU-145 but is able to inhibit urokinase-type plasminogen activator production (51). Finally, endogenous OA appears to be essential for rapid tumor cell replication and invasiveness (52). The 9c, 11t and 10t, 12c CLA isomers have been shown by other investigators to inhibit oxidation of AA by prostaglandin H synthase (53). Adding CLA to diets reduced prostaglandin F2{alpha} levels in the liver and uterus of pregnant rats and also suppressed serum proMMP-9 and active MMP-2 levels (54). In human fibroblasts, prostaglandin E2 has also been shown to stimulate and activate MMP synthesis as well as affect TIMP production (55). Therefore, a link may exist between CLA and MMP activity through alteration of eicosanoids.

It is important to understand how CLA modulates malignant tumor formation and metastasis, because secondary tumor growth is the major cause of cancer morbidity and mortality. Future studies to discover how CLA affects these and other MMP are warranted and may further elucidate the role of CLA as an inhibitor of mammary tumorigenesis.


   ACKNOWLEDGMENTS
 
We thank Ted Craig from Loders and Croklaan (Glen Ellyn, IL) for providing the CLA for these studies.


   FOOTNOTES
 
1 Supported by grant BC051055 from the Department of the Army. Back

2 Author disclosures: N. E. Hubbard, D. Lim, and K. L. Erickson, no conflicts of interest. Back

3 Abbreviations used: AA, arachidonic acid; APMA, p-aminophenyl mercuric acetate; CLA, conjugated linoleic acid; CP, crossing point; ECM, extracellular matrix; LA, linoleic acid; MMP, matrix metalloproteinase; mRNA, messenger RNA; MT, membrane type; OA, oleic acid; RCN, relative copy number; TIMP, tissue inhibitor of matrix metalloproteinase. Back

Manuscript received 6 December 2006. Initial review completed 8 January 2007. Revision accepted 30 March 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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