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,2
* Nutritional Sciences and
Cancer Biology Interdisciplinary Programs, Laboratory of Mammary Gland Biology, Department of Nutritional Sciences, The University of Arizona, Tucson, AZ 85718
2To whom correspondence should be addressed. E-mail: donato{at}u.arizona.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • conjugated linoleic acid • cell cycle arrest • G1/S • p53 • Rb
Conjugated linoleic acid (CLA)2 is a collective term for a mixture of positional and geometric isomers of linoleic acid (LA) in which the double bonds are conjugated in either the cis or trans configuration at positions 
7,9-, 8,10-, 9,11-, 10,12- or 11,13- of the carbon chain (1). CLA is found in a variety of foods including oils and seafood (0.2–0.8 mg CLA/g fat), meats (1.0–4.0 mg CLA/g fat) and dairy products (5.0–7.0 mg CLA/g fat) (2). The CLA found in ruminant meat and dairy products originates from the incomplete biohydrogenation of LA by rumen bacteria or desaturation of trans-11-octadecenoic acid by -9 desaturase (3). The cis-9, trans-11 (c9,t11-CLA) isomer is the primary dietary form of CLA in human diets. However, the relative concentration of c9,t11-CLA and other isomers including trans-10,cis-12 (t10,c12-CLA) in dairy and meat products is influenced by the type and amount of vegetable fats fed to ruminants (4). CLA inhibits chemically-induced stomach (5), mammary (6,7), colon (8) and skin (9) cancers. In vitro investigations indicate that CLA inhibits the cell growth of rat mammary organoids (10), human breast cancer cells in vitro (11,12) and chemically-induced carcinogenesis in animal models (5).
The antiproliferative effects of CLA have been attributed, at least in part, to its geometric isomerism because treatment with LA stimulates rather than represses the growth of breast (13), leukemia (14) and liver epithelial (15) cells. Studies comparing the effects of various CLA isomers suggest that t10,c12-CLA may be the more biologically active isomer for the inhibition of tumor cell proliferation (16), and elongation and desaturation of linoleic and linolenic acids (17). This cumulative evidence suggests that CLA formulations could be developed as a dietary adjuvant against neoplastic transformation (18,19). However, how CLA or specific isomers regulate the expression of cell cycle checkpoints is unknown (20).
The cell cycle progression from G0/G1 to S-phase requires phosphorylation of the retinoblastoma tumor suppressor protein, Rb, a member of the pocket protein family, by the cyclin D1-cdk4/6 and cyclin E-cdk2 complexes (21). Phosphorylation of Rb in early G1 by cyclin D1/cdk4/6 triggers a cascade of events that begins with the dissociation of E2F from Rb and the activation of transcription of cyclin E by E2F, and culminates with the stimulation by E2F of its own transcription and assembly of cyclin E with its catalytic partner Cdk2. The cyclin E-cdk2 complexes promote further phosphorylation of Rb and the release of E2F, thus establishing a positive feedback loop that accelerates the irreversible progression through late G1 (22).
The tumor suppressor p53 plays a key role in the regulation of the cell cycle in response to DNA damage and is frequently mutated in human cancers (23). The loss of p53 functions may occur early in tumorigenesis (24) or be a late event (25). The tumor suppressing properties of p53 stem from its ability to regulate, through both transcription-dependent and -independent mechanisms, the expression of a battery of genes whose products regulate cell cycle transition at the G1/S interval, or trigger apoptosis depending on the severity of DNA damage (26). In this study, we attempted to clarify the mechanisms of CLA action on the expression of restriction points controlling cell cycle transition in breast and colon cancer cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Breast (MCF-7) and colon (HCT-116) cancer cells containing wild-type p53 (p53+/+) were obtained from the American Type Culture Collection (Manassas, VA). p53-deficient (p53-/-) HCT116 cells (HCTp53KO) (obtained from Dr. Bert Vogelstein, The Johns Hopkins University School of Medicine, Baltimore, MD) were maintained in DMEM from Sigma (St. Louis, MO) supplemented with 10% fetal calf serum [(FCS) Hyclone Laboratories, Logan, UT)]. A mixture of CLA isomers (Lot #22K1142) was obtained from Sigma and consisted of t9, c11 and c9,t11 (50%), t10,c12 (40%), and c10,c12 (10%). Purified c9,t11- and t10, c12-CLA isomers were obtained from Matreya (State College, PA). The effects of the CLA preparations on cell proliferation were investigated using the trypan blue exclusion and methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell proliferation assays. The trypan blue assay protocol was as previously described (27), and is based on the exclusion of the trypan blue dye by viable cells and uptake by dead cells. Briefly, cells were seeded in quadruplicate at a density of 0.5 x 106 cells/6-well tissue culture plates and maintained in DMEM plus 10% FCS. At the end of the incubation periods, cells were washed with PBS, trypsinized and counted using a hemocytometer. The MTT proliferation assay kit was obtained from Promega (Madison, WI). This assay is based on the conversion of the yellow tretrazolium dye MTT to purple formazan crystals by metabolically active cells (28). Briefly, cells were seeded in 96-well tissue culture plates and maintained overnight in DMEM plus 10% FCS. Four wells were assigned to each experimental treatment. Cells were treated with various concentrations of CLA or specific isomers for various periods of time. At 24, 48 or 72 h after treatment, 15 µL of MTT dye solution was added to each well, and the plate was incubated for 4 h at 37°C. Solubilization/stop solution (100 µL) was added for 1 h at 37°C and the absorbance at 570 nm was recorded using a Synergy HT plate reader (Bio-Tek Instruments, Winooski, VT). Flow cytometry was performed in triplicate as described previously (29). Briefly, cells were harvested with trypsin and washed in PBS. Cells were then treated with RNAse and stained with propidium iodide (70 µmol/L in PBS). Cell cycle distribution profiles were recorded with a FACscan (Becton-Dickinson, San Diego, CA), using a CELLQuest program.
Transient transfections and Western blot analysis.
Transient transfections were performed using the Lipofectamine-Plus procedure according to manufacturer’s instruction (Life Technologies, Gaithersburg, MD) and as described previously (27). Cells were transfected for 3 h with 3 µg of the expression vectors pCMV (empty) or pCMV53mut containing a cassette encoding for p53 mutated at position 175 (Arg to His) under the control of the cytomegalovirus promoter (CVM) (plasmids were gifts from Dr. Bert Vogelstein). The transfected cells were cultured in the absence or presence of 160 µmol/L CLA for 24 h prior to the harvesting of cell lysates. Western blotting was performed as described previously (27). Cell extracts were normalized to protein content and separated by 4 to 12% gradient SDS-PAGE. Immunoblotting was carried out with antibodies raised against p53, p21, p27 (Oncogene Research Products, Cambridge, MA), cyclin D1 and cyclin E (BD Pharmagen, San Diego, CA). The effects of CLA on the phopshorylation status of Rb were investigated using antibody mAb245 (BD Pharmagen, San Diego, CA) that recognizes both hypo- and hyperphosphorylated Rb (
110–116 kDa) and mAb549, which recognizes only the fast migrating 110 kDa underphosphorylated Rb protein (Biomol Research Laboratories, Plymouth Meeting, PA). The normalization of Western blots was confirmed by incubating immunoblots with ß-actin antibody-1 (Oncogene Research Products). The immunocomplexes were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Data collection and statistical analysis.
Cell viability and flow cytometry data are presented as means ± SEM. The comparison of means following a significant (P < 0.05) ANOVA were performed by the Fisher protected least significant difference test. Flow cytometry data were analyzed with the MODFIT.2 software at the Flow Cytometry Laboratory, Arizona Cancer Center.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The effects of a mixture of CLA isomers on cell proliferation of asynchronous MCF-7 cells were investigated using the trypan blue exclusion assay. This assay provides a direct count of viable cells that exclude the trypan blue dye. The treatment with CLA for 72 h resulted in a 20% inhibition of cell proliferation starting at concentrations of 40 µmol/L (Fig. 1A), whereas 160 µmol/L CLA reduced the growth rate by 45%. The antiproliferative effects of CLA on MCF-7 cells were further examined using the MTT proliferation assay, which provides an indirect but quantitative determination of metabolically active cells (28). Compared with cells cultured in control medium, the treatment with the CLA mixture for 72 h resulted in a 20 to 30% reduction in cell viability with a concentration range of 10–80 µmol/L, and a 50% reduction in the presence of 160 µmol/L CLA (Fig. 1B). Therefore, compared with the trypan blue method, the MTT assay was more sensitive, indicating that at concentrations as low as 10 µmol/L the CLA treatment reduced the number of metabolically active cells.
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41.0%) and S-phase (43.0%) windows. Conversely, the treatment with CLA for 24 h delayed MCF-7 cells in G0/G1 (53.4%) while reducing the accumulation in the S-phase (28.7%), but had no effect on the fraction of cells occupying the G2/M window (17.8 versus 16.6%) (Fig. 2A). Western blot analysis of cell lysates obtained from MCF-7 cells indicated that the CLA-induced arrest in G0/G1 was paralleled by the accumulation of p53 and p21 proteins (Fig. 2B). In addition, the CLA mixture increased the levels of hypophosphorylated Rb, as evidenced by the appearance of a doublet comprising a faster migrating band (110 kDa). The Rb protein migrates on an SDS-PAGE as multiple closely spaced bands (110–116 kDa). The different bands represent different Rb phosphorylation states, which are cell cycle dependent and were detected in this study using antibody mAb245 that recognizes both hypo- and hyperphosphorylated Rb. In MCF-7 cells cultured in control medium (DMEM), we observed the accumulation of cyclin D1 (6 to 24 h) and cyclin E (18 h). In contrast, in CLA-treated cells we observed a reduction of cyclin D1 (6 to 24 h) and E (6 and 18 h) (Fig. 2C) protein levels. Both cyclin D1 and cyclin E are required for progression to S-phase and their reduced expression in CLA-treated cells corroborated the flow cytometry data (Fig. 2A) documenting the arrest of MCF-7 cells in G1 phase of the cell cycle following treatment with the CLA mixture.
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The kinetics of cell cycle arrest induced by the CLA mixture were further investigated by first synchronizing MCF-7 cells in G0/G1 (Fig. 3). At the end of the synchronization period, 94% of the cells were positioned in G0/G1. The cells were then released in control DMEM or DMEM plus 160 µmol/L CLA and their position in the cell cycle was examined by flow cytometry. The treatment with the CLA mixture for 12 h did not influence cell cycle distribution (Fig. 3A–C) or the cellular contents of p53, p21 and Rb status irrespective of the presence or absence of CLA in the culture media (data not shown). However, the treatment with CLA for 24 h increased the percentage of cells delayed in G0/G1 (68 versus 53%), while reducing the fraction of cells positioned in S-phase (30 versus 45%) and G2/M (1.9 versus 6.5%). These data provide important evidence that the CLA mixture induced cell cycle arrest in G1. This effect was accompanied by the accumulation of p53, p21 and the faster migrating immunocomplex representing hypophosporylated Rb (Fig. 3D). Based on these results, we hypothesized that one mechanism by which CLA may induce G1 arrest is through stabilization of p53, which in turn initiates a cascade of events including the activation of p21 expression and reduction of cyclin E expression. To test this hypothesis, MCF-7 cells were transfected with an expression vector (p53mut) containing a cassette encoding for p53 mutated at position 175 (Arg to His) under the control of the cytomegalovirus (CMV) promoter. After transfection, cells were cultured for 48 h in the presence (160 µmol/L) or absence of CLA and cell lysates were analyzed by Western blotting. The data (Fig. 4) indicate that in MCF-7 cells transfected with the empty vector (pCMV) the treatment with the CLA mixture led to the accumulation of p53 and p21 proteins, while reducing the levels of immunocomplexes representing cyclin E. As a result of constitutive activity from the CMV promoter, transfection with the p53mut vector increased the intensity of immunocomplexes for p53 irrespective of the absence or presence of CLA in the culture media. More importantly, the overexpression of mutant p53 (p53mut) prevented the accumulation of p21 and the reduction of cyclin E observed in MCF-7 cells transfected with pCMV. These observations confirm that the suppressive effects of CLA on cyclin E levels were mediated by p53.
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To further investigate the role of p53 in the CLA-induced arrest in G1, the effects of CLA on G1 checkpoints in colon cancer HCT116 cells containing wild-type p53+/+ and p53-deficient HCT116 cells (HCTp53KO) were examined. Western blot analysis indicated that the treatment of HCT116 cells with CLA (160 µmol/L) exerted effects similar to those seen in MCF-7 cells including the accumulation of p53, p21, p27 and hypophosphorylated Rb (pRb) (Fig. 5A). Immunoblotting of cell lysates obtained from MCF-7 and HCT116 cells with the antibody, mAb549, which recognizes only hypophosphorylated Rb, provided direct evidence that the treatment with the CLA mixture caused accumulation of pRb and that this effect was p53-dependent. In fact, CLA failed to change the levels of p27 and Rb phosphorylation status in p53-deficient HCTp53KO cells. However, in keeping with earlier reports documenting the induction of p21 through p53-independent mechanisms (32), a slight accumulation of p21 in HCTp53KO cells treated with CLA was observed. Time-course experiments with HCT116 cells (p53+/+) confirmed that CLA induced the stabilization of p53, p27 and p21 while reducing from 6 to 18 h the expression of cyclin E (Fig. 5B). Conversely, no time-dependent changes in the cellular content of p27 and Rb in HCTp53KO cells treated with CLA were observed (data not shown). These results provide direct evidence that the reduction of hyperphosphorylated retinoblastoma (ppRb) protein levels observed in cells treated with a mixture of CLA isomers results from the activation of p53-dependent mechanisms.
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To determine the physiological importance of the CLA levels used in these studies and which CLA isomer was responsible for altering p53 protein and Rb phosphorylation status, the effects of various concentrations of the CLA mixture with the c9, t11-CLA and t10,c12-CLA isomers were compared. The MTT proliferation data from 24 to 72 h (Fig. 6) indicated that the c9,t11-CLA isomer induced a modest 5% decrease in cell number at concentrations ranging from 10 to 40 µmol/L (Fig. 6B). At 24 h, the treatment with 80 and 160 µmol/L c9,t11-CLA reduced cell number by 20%. Conversely, the treatment with t10,c12-CLA at concentrations ranging from 10 to 40 µmol/L CLA as early as 24 h produced a larger reduction (25%) in cell viability that was further reduced to 35 and 55%, respectively, after treatment with 80 and 160 µmol/L CLA for 72 h (Fig. 6C). The CLA mixture and the t10,c12-CLA isomer produced similar growth inhibition. At the concentration of 40 µmol/L, the CLA mixture and t10,c12-CLA, but not c9,t11-CLA, were equally effective in inducing the accumulation of p53 protein (Fig. 6D). Titration experiments confirmed that at concentrations as low as 10 µmol/L, the t10,c12-CLA isomer was more effective than c9,t11-CLA in increasing p53 (Fig. 7A) and pRb (Fig. 7B) levels. These data support the conclusion that the t10,c12-CLA isomer is more effective than c9,t11-CLA in enhancing an antiproliferative response by lowering the expression of factors (ppRb) required for transition through G1.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To examine the contribution of p53 to CLA-induced growth arrest, a mutant p53 (175Arg to His) was overexpressed in MCF-7 cells, which prevented the accumulation of p21 and the reduction in cyclin E seen in nontransfected MCF-7 cells treated with CLA. These data provide important mechanistic evidence that the activation of p53-regulated pathways may be central to the antiproliferative effects of CLA. The p53 mutant 175Arg to His, which is comprised in the DNA-binding domain of the p53 protein, was used for the transfection experiments because it is the most frequently mutated p53 codon in breast and colon tumors. The notion that p53 plays a key role in the CLA-induced cell cycle arrest was further corroborated by the observation that the treatment of colon cancer HCTKO (p53-/-) cells with CLA failed to elicit any changes in the expression of p27 or the relative ratio of ppRb/pRb.
These results suggest that the health effects of CLA may be mediated by the expression of wild-type p53. The p53 gene product is known to be a key player in the genotoxic-stress response in mammalian cells by inducing the transcription of p21, which in turn inhibits cyclin E/cdk2-dependent phosphorylation of Rb, thus leading to the accumulation of pRb (21). The transition from G1 to S-phase requires phosphorylation of the retinoblastoma tumor suppressor protein Rb by the cyclin D1-cdk4/6 (G0 to mid-G1) and cyclin E-cdk2 (late G1) complexes. The phosphorylation of Rb in early G1 leads to the dissociation of E2F transcription factors and the transcriptional activation of genes, the protein products of which are required for the transition through late G1 including cyclin E and E2F1 (22). In contrast, the cdk inhibitors p21 and p27 stechiometrically regulate the activities of cyclin D1-Cdk4/6 and cyclin E-Cdk2 complexes during the G1 phase of the cell cycle (36). In proliferating cells, the p21 protein facilitates the assembly of the cyclin D1-Cdk4 complexes, whereas a reduction in the levels of cyclin D1 increases the pool of available p21, which then associates with cyclin E-cdk2 complexes, thus reducing cyclin E-cdk2-dependent kinase activity (37). Similarly, p27 outtiters cyclin D1 thus inhibiting cyclin D1-dependent activation of Cdk4/6 (38). These overall findings suggest that by triggering the p53-dependent accumulation of p21 and p27, CLA causes a reduction in the cellular levels of cyclin E and ppRb leading to G1 arrest.
Previous studies have suggested that the biological effects of CLA mixtures are, at least in part, due to the distinct actions of the c9,t11- and t10,c12-isomers (4). Therefore, we examined the effects of various concentrations of these CLA isomers on cell proliferation and expression of p53 and Rb proteins. Our results indicate that there are marked differences in the antiproliferative activity of c9, t11-CLA and t10,c12-CLA. At concentrations as low as 10 µmol/L, which approximate CLA plasma levels measured in humans (39,40), the t10,c12-CLA isomer was more potent than c9,t11-CLA in repressing cell growth and inducing the accumulation of p53 and pRb in breast cancer MCF-7 cells. Thus, the relative concentration of the t10,c12-CLA isomer may influence the health effects of CLA mixtures found in enriched preparations, and meat and dairy products.
The increase in the steady-state levels of p53 observed in MCF-7 cells treated with t10,c12-CLA suggests that its growth inhibitory properties may involve the induction of a DNA damage response. Previous studies showed that CLA induces lipid peroxidation in breast MCF-7 and colon SW480 cancer cells (41), and modulates both nonenzymatic arachidonic acid oxidation and cyclooxygenase-catalyzed prostaglandin profile (42). These effects of CLA on lipid metabolism may be exerted through the modulation of PPAR as evidenced by the fact that CLA is a ligand of both PPAR
(43) and PPAR-
(44), and PPAR are involved in the transcriptional regulation of the cyclooxygenase-2 gene (45). Therefore, it is plausible that the ability of specific CLA isomers or metabolic derivatives to elicit changes in oxidative damage may contribute to the activation of p53-dependent pathways leading to cell cycle arrest or apoptosis depending on the intensity and duration of exposure. Although the regulation of apoptosis by CLA was not the primary focus of this study, an increase in the mRNA levels for Bax- and a reduction in Bcl-2 expression in MCF-7 cells treated with the CLA mixture was observed (Kemp, M. Q., Jeffy, B. D. & Romagnolo, D. F., unpublished data). These observations corroborate earlier reports documenting the induction of apoptosis by CLA in MNU-initiated rat mammary gland (46). Similar growth inhibitory properties have been described for docosahexaenoic acid [22:6(n-3)], which decreases Rb phosphorylation in melanoma SK-Mel-110 cells (47) and increased cytotoxicity induced by doxorubicin (48). The implication of p53 in the regulation of the expression of proapoptotic and antiapoptotic genes suggests that the loss of functional p53 may confer resistance to CLA treatment.
In summary, this report documents that in breast and colon cancer cells, CLA compromises the expression of cyclins required for G1/S progression including cyclin D1 and E, and the phosphorylation status of Rb. This cell cycle regulatory aspect of CLA may have clinical importance in the development of preventive and/or therapeutic strategies based on the fact that nearly 45% of breast cancers overexpress cyclin D1 (49), and overexpression of either cyclin D1 or cyclin E contributes to neoplastic progression of the breast (50) and colon (51). Although further studies are required to clarify whether CLA-induced p53 and related withdrawal from the cell cycle may be due to altered lipid peroxidation and/or generation or reactive metabolites, our findings document for the first time that the effects of CLA on the expression of G1 checkpoints may depend on the expression of functional p53. However, the fact that p53 is the most commonly mutated gene in human cancers may limit the usefulness of therapeutic strategies based on CLA. Finally, whereas our data do not preclude the possibility that the t10,c12- and c9,t11-CLA or other isomers may act synergistically to inhibit cell proliferation, this study attributes a major suppressive effect on the cell cycle apparatus to the t10,c12-CLA isomer. The molecular mechanisms through which this and other CLA isomers may alter the components of the cell cycle await further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CLA, conjugated linoleic acid; CMV, cytomegalovirus; FCS, fetal calf serum; HCTKO, HCT116 cells (p53-/-); LA, linoleic acid; MTT, methylthiazolyldiphenyl-tetrazolium bromide; p53+/+, wild-type p53; PPAR, peroxisome proliferator-activated receptor; ppRb, hyperphosphorylated retinoblastoma; pRb, hypophosphorylated retinoblastoma.
Manuscript received 8 May 2003. Initial review completed 10 June 2003. Revision accepted 28 August 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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