Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth

Eun J. Kim1, P. Elly Holthuizen2, Hyun S. Park3, Yeong L. Ha4, Kyeong C. Jung5, and Jung H. Y. Park1

1 Division of Life Sciences and 5 Department of Pathology, Hallym University, Chunchon 200-702; 3 Department of Food and Nutrition, Kyung Hee University, Seoul 130-701; and 4 Division of Applied Life Sciences, Graduate School, Gyeongsang National University, Chinju 660-701, Korea; and 2 Laboratory for Physiological Chemistry, University Medical Center Utrecht, 3584 CG Utrecht, Netherlands


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 ± 3% reduction 96 h after adding 5 µM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (Mr) IGF-II and higher Mr forms of IGF-II. The levels of the higher Mr and the mature form of IGF-II were decreased 50 ± 3% and 22 ± 2%, respectively, by 5 µM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.

insulin-like growth factor binding proteins; apoptosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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THE INCIDENCE OF COLORECTAL cancer, one of the most prevalent cancers in the Western world, is increasing, and there is increasing urgency to develop strategies to prevent this disease. With regard to prevention, diet (37) has drawn considerable attention in recent years.

Conjugated linoleic acid (CLA) is a naturally occurring substance in food sources such as the fat of milk and meat of ruminant animals (23). CLA consists of eight possible geometric isomers of 9,11- and 10,12-octadecadienoic acid with cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12) CLA being the principal isomers (32). Blood levels of CLA isomers in nonvegetarians have been reported (12) to be in the range of 20-70 µM, with the c9t11 and t10c12 isomers representing ~80% and ~10%, respectively. CLA has been shown to have a number of biological actions. Animal studies (2, 3, 15, 17, 24, 33) have demonstrated that CLA inhibits tumorigenesis in mammary, forestomach, colon, and skin models of carcinogenesis in rats. In addition to animal studies, in vitro studies have shown that CLA inhibits the growth of human colon cancer cells, SW 480 (30) and HT-29 cells (40). These studies (2, 3, 15, 17, 24, 30, 33, 40) have used CLA preparations containing mixtures of CLA isomers; therefore, the effect of individual CLA isomers on colon cancer cell proliferation and the mechanisms by which they influence cancer cell proliferation remain unclear.

Several peptide growth factors have been shown to play an important role in regulating normal cell growth, and abnormal expression of these growth factors has been implicated in the etiology of cancer. The insulin-like growth factors (IGFs) are single-chain polypeptides with structural homology to proinsulin and are powerful mitogens for a variety of mammalian cells. Most of the cellular effects of IGFs are mediated by the type I IGF receptor (IGF-I), and IGF actions are modulated by interactions with IGF-binding proteins (IGFBPs) (20, 44). Six high-affinity IGFBPs, IGFBP-1 through IGFBP-6, have been identified (1), and four IGFBP-related proteins have been found to have a relatively low affinity for IGFs (16). IGFBPs modulate IGF effects in metabolic regulation, cell growth, and tumorigenesis (48). IGFBPs also have IGF-independent effects, exerting direct cellular actions independent of IGF-I (1).

Several elements of the IGF axis exhibit altered expression and thereby may play an important role in the development of colon cancer (42). In human colon carcinoma, IGF-II mRNA (51) and IGFBP-2 mRNA (27) were overexpressed compared with normal adjacent tissues, and circulating IGF-II and IGFBP-2 were elevated (38). Colon tumor tissues and tumor cell lines have higher IGF-I receptor numbers compared with normal mucosa (13). The human colon adenocarcinoma cell line Caco-2, which is used in the present study, synthesizes and secretes IGF-II, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 (22, 34, 52), and IGF-II acts as an autocrine growth regulator for these cells (49, 50).

The present study was performed to determine which of the two CLA isomers (t10c12 or c9t11) inhibits Caco-2 growth and to assess whether such an effect is related to changes in the secretion of IGF-II and/or IGFBPs that have been shown to regulate Caco-2 cell proliferation. Our results demonstrate that t10c12 CLA is the isomer responsible for the inhibition of Caco-2 cell growth.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Materials. Caco-2 cells (HTB-37, passage 23) were obtained from American Type Culture Collection (Rockville, MD). DMEM/Ham's F-12 nutrient mixture (DMEM/F12), essentially fatty acid-free BSA, linoleic acid (LA), ascorbic acid, alpha -tocopherol phosphate, a mixture of CLA isomers (31), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS), trypsin-EDTA, penicillin-streptomycin, transferrin, and selenium were obtained from GIBCO-BRL (Gaithersburg, MD). Cell culture plastics were purchased from Corning (Corning, NY), and nylon membranes were obtained from Micron Separations (Westborough, MA). A monoclonal antibody specific for IGF-II (45) was purchased from Amano International Enzyme (Troy, VA). Nitrocellulose transfer membranes (BA 83) were obtained from Schleicher and Schuell (Keene, NH). The enhanced chemiluminescence system, rainbow molecular weight (Mr) markers, anti-rabbit and anti-mouse secondary antibodies, [methyl-3H]thymidine, and (3-[125I]iodotyrosyl)IGF-II (specific activity 2,000 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). C9t11 and t10c12 CLA were obtained from Cayman Chemical (Ann Arbor, MI). Recombinant human IGF-II was donated by M. H. Niedenthal of Lilly Research Laboratories (Indianapolis, IN).

Cell culture. Caco-2 cells between the 28th and 32nd passage were used for these experiments. Cells were maintained and subcultured at 37°C in a humidified CO2 incubator (95% air-5% CO2). The complete medium for cell maintenance consisted of DMEM/F12 containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. When cells were 80% confluent, they were subcultured using 0.25% trypsin-2.65 mM EDTA, and the medium was changed every 2 days. All cultures contained ascorbic acid (50 ng/ml) and alpha -tocopherol phosphate (20 ng/ml) to protect fatty acids from peroxidation. To examine the effects of the CLA isomers, we rinsed and serum starved cell monolayers in DMEM/F12 supplemented with 5 µg/ml transferrin, 5 ng/ml selenium, and 0.1 mg/ml BSA. After serum starvation, cells were incubated in the presence or absence of various concentrations of t10c12 or c9t11 CLA. Fatty acids were complexed to essentially fatty acid-free BSA, with the molar ratio of fatty acid to BSA being 4:1, as described previously (22). Medium was changed every 2 days. The basal serum-free medium containing 0.15 µM LA was chosen to eliminate the possibility of an essential fatty acid deficiency. Viable cell numbers were estimated by the MTT assay (9). To study the effects of fatty acids on cell proliferation, the cell monolayers were similarly treated with individual fatty acids and [3H]thymidine incorporation was determined as described previously (35).

Ligand blot, dot blot, and immunoblot analysis. Caco-2 cells were cultured as described above. Twenty four-hour conditioned medium was collected between days 2 and 3 of culture, centrifuged at 3,000 g for 10 min to remove cell debris, and stored at -20°C in the presence of 1 mM phenylmethylsulfonyl fluoride. The conditioned medium was concentrated 10-fold using microconcentrators with a Mr exclusion of 3,000 (Amicon, Danvers, MA). The retentates were used for ligand blot analysis of IGFBPs and immunoblot analysis of IGF-II. For the determination of IGFBPs, the proteins of the concentrated medium were analyzed by 10-13% gradient SDS-PAGE, followed by electrotransfer to BA83 nitrocellulose paper. The blots were probed with 125I-labeled IGF-II as described previously (21). For IGF-II determination, the proteins were separated on 10-20% gradient SDS-PAGE and electrotransferred to BA83 paper. The blots were probed with 10 ng/ml of a mouse monoclonal antibody against rat IGF-II, a sheep anti-mouse biotinylated IgG, and streptoavidin-biotinylated horseradish peroxidase complex as described by Kim et al. (21). To achieve the better quantitation of total IGF-II produced by Caco-2 cells, we serially diluted and analyzed the conditioned medium by dot blot analysis as described by De Leon et al. (7). The bands and dots were visualized using the enhanced chemiluminescence detection system. The relative abundance of each band was measured by a densitometric scanning of the exposed films using a densitometer (Molecular Dynamics, Sunnyvale, CA).

Northern blot analysis. Caco-2 cells were seeded at the density of 6 × 105 cells/dish in 100-mm dishes and cultured in DMEM/F12 supplemented with 10% FBS. After 24 h, monolayers were rinsed twice with DMEM/F12 and incubated for 3 days in serum-free medium in the absence (control) or presence of various concentrations of individual fatty acids. Total RNA was isolated using Tri-Reagent (Sigma Chemical), according to the method of Chomczynski (5), and fractionated on 1% agarose-formaldehyde gels. RNA was then transferred onto a positively charged membrane by capillary transfer, cross-linked to the membrane using a Stratalinker (Stratagene, La Jolla, CA), and probed with an antisense IGF-II cRNA. A human 980-bp IGF-II cDNA (19) was linearized utilizing Sal I, and a cRNA probe was synthesized using digoxigenin-UTP (Roche Molecular Biochemicals, Mannheim, Germany) by in vitro transcription utilizing T3 polymerase. DIG luminescent detection kit (Roche Molecular Biochemicals) was used to identify mRNA species visualized by exposing the membrane to an X-ray film. As a control for RNA levels and integrity, Northern blot analyses were also performed with an antisense cRNA probe of human cyclophilin (pTRI-cyclophilin-human, Ambion, Austin, TX). The relative abundance of each band was estimated by densitometric scanning of the exposed films.

DNA laddering. Caco-2 cells were cultured and serum starved as described above. After serum starvation, the monolayers were incubated with or without t10c12 CLA in the absence or presence of 200 nM IGF-II. Cells were extracted for 2 h in extraction buffer (50 mM Tris, pH 7.5, 20 mM EDTA, and 1% Nonidet P-40). SDS was then added to 1%, and the mixture was incubated for 2 h with 500 µg/ml RNase at 37°C followed by an incubation for 2 h with 500 µg/ml proteinase K at 42°C. The mixture was then extracted with phenol-chloroform-isoamylalcohol (25:24:1), and the DNA was precipitated with 0.3 M sodium acetate and 2.5 vol of absolute ethanol. Equal amounts of DNA samples (20 µg) were electrophoresed on a 2% agarose gel in Tris-borate EDTA buffer and visualized by ethidium bromide staining.

Fluorescence-activated cell-sorting analysis. To estimate apoptotic cell number, cells were plated in 24-well plates and incubated in the absence or presence of t10c12 and/or IGF-II. After 3 days, cells were trypsinized and incubated with phycoerythrin (PE)-conjugated annexin V (annexin V-PE) and 7-aminoactinomycin D (7-AAD) (BD Pharmingen, Franklin Lakes, NJ) for 15 min at room temperature in the dark. Apoptotic cells were analyzed by flow cytometry within 1 h utilizing FACScan (Becton Dickinson, Franklin Lake, NJ).

Statistical analysis. Data are expressed as means ± SE. Differences among treatment groups were analyzed by ANOVA and Duncan's multiple range test (43) utilizing Statistical Analysis Systems statistical software package version 6.12 (SAS Institute, Cary, NC).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

t10c12 CLA decreased Caco-2 cell growth in dose-dependent manner. To determine which CLA isomer is more efficacious in inhibiting Caco-2 cell growth, we incubated cells in serum-free medium in the absence or presence of various concentrations of t10c12 or c9t11 CLA. As depicted in Fig. 1, t10c12 CLA decreased viable cell numbers in a dose-dependent manner with a 55 ± 3% decrease in cell number within 96 h after the addition of 5 µM t10c12 CLA (Fig. 1A). These effects were specific for t10c12 CLA, because the inclusion of the same concentrations of c9t11 CLA in the incubation medium had no effect (Fig. 1B). To compare the long-term effects of the CLA mixture and the two isomers on Caco-2 cell growth, cells were cultured for 12 days in serum-free medium in the absence (control) or presence of 2.5 µM of the CLA mixture, t10c12 CLA, or c9t11 CLA. As shown in Fig. 2, Caco-2 cells grew well in serum-free medium, reaching a plateau density at 8 days of culture, and the CLA mixture significantly inhibited cell growth at 2.5 µM. Doubling times under these conditions were calculated from the linear part of the growth curves. The doubling times of CLA mixture-treated cells and control cells were calculated to be 30 ± 1 and 24 ± 1 h, respectively (P < 0.05). The cells did not grow in medium containing 2.5 µM t10c12 CLA after 4 days of the fatty acid treatment. However, the same concentration of c9t11 CLA did not have any effect on the growth patterns of these cells. Because the MTT assay measured the number of viable cells, we next determined whether the CLA isomers inhibit cell proliferation utilizing the [3H]thymidine incorporation assay. The results of the [3H]thymidine incorporation assay are shown in Fig. 3. DNA synthesis was reduced by the t10c12 isomer in a dose-dependent manner. However, the degree of the decrease in [3H]thymidine incorporation was smaller than that of viable cell numbers determined by the MTT assay, indicating that the decrease in viable cell number by CLA is partially due to decreased DNA synthesis. In fact, the differences between 0 and 1 µM t10c12 CLA, 1 and 2.5 µM t10c12 CLA, and 2.5 and 5 µM t10c12 CLA were not statistically significant (P < 0.05). The c9t11 isomer did not have any effect on DNA synthesis of Caco-2 cells.


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Fig. 1.   Effect of trans-10,cis-12 (t10c12; A) and cis-9,trans-11 (c9t11; B) conjugated linoleic acid (CLA) on Caco-2 cell growth. Caco-2 cells were plated in 24-well plates at 40,000 cells/well in DMEM/Ham's F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS). One day later, the monolayers were serum starved with serum-free DMEM/F12 supplemented with 5 µg/ml transferrin, 0.1 mg/ml BSA, and 5 ng/ml selenium for 24 h. After serum starvation, cells were incubated in serum-free medium in the absence or presence of various concentrations of t10c12 or c9t11. Cell numbers were estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Values are means ± SE; n = 6. ANOVA followed by Duncan's multiple range test at each time point was used to determine significant differences among the treatment groups. Values with different letters are significantly different (P < 0.05). Values with the same letter are not significantly different.



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Fig. 2.   Caco-2 cell growth curves. Cells were seeded at a density of 30,000 cells/well in 24-well plates and serum starved as described in the Fig. 1 legend. After serum starvation (day 0), cells were continuously grown in serum-free medium in the absence (control) or presence of 2.5 µM c9t11, t10c12, or a mixture of CLA isomers (CLA), and the medium was replaced every 2 days. Cell numbers were determined by the MTT assay. Values are means ± SE from 6 independent experiments.



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Fig. 3.   Effect of t10c12 and c9t11 CLA on [3H]thymidine incorporation in Caco-2 cells. Caco-2 cells were plated in 96-well plates at 5,000 cells/well and treated with t10c12 or c9t11 CLA for 3 days as described in the Fig. 1 legend. [3H]thymidine was then added, and the incubation was continued for another 14 h to measure the incorporation into DNA. Values are means ± SE; n = 6. Values with different letters are significantly different (P < 0.05) between concentrations of individual fatty acids. Values with the same letter are not significantly different.

t10c12 CLA inhibited IGF-II production and IGF-II mRNA expression in Caco-2 cells. Caco-2 cells have been shown (50) to maintain an autocrine growth loop driven by endogenous expression of IGF-II, which may be able to sustain proliferation in the absence of serum, as observed in the experiment depicted in Fig. 2. To determine whether the CLA isomers decrease IGF-II secretion, 24-h serum-free conditioned medium was collected and immunoblot analyses were performed with a monoclonal antibody against rat IGF-II (45). Immunoblot analysis revealed that Caco-2 cells secrete two different immunoreactive IGF-II-like species; a band with an apparent Mr of 7,500 and a doublet of bands with Mr of 11,000 and 14,300 (Fig. 4A). The majority of IGF-II was present as the high-Mr forms, which may be pro-IGF-II (6). Treatment with t10c12 CLA decreased the intensity of the high-Mr bands in a dose-dependent manner. Levels of the low-Mr form, representing the mature IGF-II, were also decreased dose dependently by treatment with t10c12 CLA. The degree of the decrease was smaller with the low-Mr form vs. the high-Mr band (Fig. 4B). C9t11 CLA did not affect production of either the high-Mr or low-Mr form of IGF-II (Fig. 5). To achieve more reliable quantification of total IGF-II, we performed dot blot analyses. However, the results were similar to those observed with Western blot analyses showing that the t10c12 isomer inhibited IGF-II production in a concentration-dependent manner whereas the c9t11 isomer had no effect (data not shown).


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Fig. 4.   Effect of t10c12 CLA on insulin-like growth factor II (IGF-II) secretion by Caco-2 cells. Cells were plated and cultured as described in the Fig. 1 legend. After serum starvation, the monolayers were incubated in serum-free medium in the absence or presence of various concentrations of t10c12. Twenty four-hour conditioned medium was collected and concentrated for immunoblot analysis with a monoclonal antibody against IGF-II. The volumes of medium loaded onto the gel were adjusted for equivalent cell numbers. A: a photograph of chemiluminescent detection of a blot, which is representative of 3 independent experiments. Mr, molecular weight; STD, standard. B: quantitative analysis of immunoblots. Values are means ± SE; n = 3. Values with different letters are significantly different (P < 0.05) within pro-IGF-I and IGF-I groups. Values with the same letter are not significantly different.



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Fig. 5.   Effect of c9t11 CLA on IGF-II secretion by Caco-2 cells. Cells were plated and cultured as described in the Fig. 1 legend. After serum starvation, the monolayers were incubated in serum-free medium in the absence or presence of various concentrations of c9t11. Twenty four-hour conditioned medium was collected and concentrated for immunoblot analysis with a monoclonal antibody against IGF-II. The volumes of medium loaded onto the gel were adjusted for equivalent cell numbers. A: a photograph of chemiluminescent detection of a blot, which is representative of 3 independent experiments. B: quantitative analysis of immunoblots. Values are means ± SE; n = 3. Values with the same letter are not significantly different within pro-IGF-I and IGF-I groups.

To determine whether the decreased IGF-II protein concentrations are related to the steady-state levels of mRNA, we performed Northern blot analyses. Northern blot analysis of total RNA using a human IGF-II cRNA probe revealed two bands exhibiting apparent sizes of 6 and 4.8 kb. The 6- and 4.8-kb IGF-II mRNA species decreased after treatment with 5 µM t10c12 CLA. The decrease in the 6-kb mRNA species was much smaller than that in the 4.8-kb species (Fig. 6). However, the same concentration of c9t11 CLA increased the 6-kb species of IGF-II mRNA but did not alter the 4.8-kb species (Fig. 7).


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Fig. 6.   Effect of t10c12 CLA on IGF-II mRNA levels in Caco-2 cells. Cells were seeded at a density of 6 × 105 cells/dish in 100-mm dishes with DMEM/F12 supplemented with 10% FBS. After 24 h, the monolayers were rinsed with serum-free medium and serum starved for 24 h. Cells were then incubated in serum-free medium with various concentrations of t10c12 CLA. A: total RNA was isolated for Northern blot analysis probing with IGF-II cRNA (top) or human cyclophilin cRNA (bottom). Photographs of chemiluminescent detection of the blots are representative of 3 independent experiments. B: relative abundance of each IGF-II band relative to its cyclophilin control band on Northern blot was estimated by densitometric analysis. Values are means ± SE from 3 independent experiments. Values with different letters are significantly different (P < 0.05) within 6- and 4.8-kb groups. Values with the same letter are not significantly different.



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Fig. 7.   Effect of c9t11 CLA on IGF-II mRNA levels in Caco-2 cells. Cells were seeded and serum starved as described in the Fig. 6 legend. After serum starvation, cells were incubated in serum-free medium with various concentrations of c9t11. A: total RNA was isolated for Northern blot analysis and probed with IGF-II cRNA (top) or human cyclophilin cRNA (bottom). Photographs of chemiluminescent detection of the blots are representative of 3 independent experiments. B: relative abundance of each IGF-II band relative to its cyclophilin control band on Northern blot was estimated by densitometric analysis. Values are means ± SE from 3 independent experiments. Values with different letters are significantly different (P < 0.05) within 6- and 4.8-kb groups. Values with the same letter are not significantly different.

To examine whether the t10c12 CLA treatment causes any changes in IGFBP concentrations, we collected 24-h conditioned medium by incubating cells in serum-free medium in the absence or presence of various concentrations of t10c12 CLA. Aliquots (300 µl) of conditioned medium were concentrated 10-fold by ultrafiltration, and the concentrates were analyzed by ligand blot analysis (Fig. 8A). As previously reported, Caco-2 cells produced three IGFBPs: 34,000-Mr IGFBP-2 (34), 24,000-Mr IGFBP-4 (34), and 30,000-Mr IGFBP-6 (21, 52). The concentrations of all three IGFBPs were slightly decreased by 5 µM t10c12 CLA (both P < 0.05 vs. control, Fig. 8), whereas the c9t11 isomer had no effect on the concentrations of IGFBPs in the conditioned medium (Fig. 9).


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Fig. 8.   Effect of t10c12 CLA on IGF-binding protein (IGFBP) secretion by Caco-2 cells. Cells were plated and cultured as described in the Fig. 1 legend. After serum starvation, the monolayers were incubated in serum-free medium in the absence or presence of various concentrations of t10c12 CLA. Twenty four-hour conditioned medium was collected and concentrated for ligand blot analysis utilizing 125I-labeled IGF-II. A: the autoradiograph of a ligand blot, which is representative of 3 independent experiments. B: quantitative analysis of ligand blots. Values are means ± SE; n = 3. Values with different letters are significantly different (P < 0.05) within IGFBP groups. Values with the same letter are not significantly different.



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Fig. 9.   Effect of c9t11 CLA on IGFBP secretion by Caco-2 cells. Cells were plated and cultured as described in the Fig. 1 legend. After serum starvation, the monolayers were incubated in serum-free medium in the absence or presence of various concentrations of c9t11 CLA. Twenty four-hour conditioned medium was collected and concentrated for ligand blot analysis utilizing 125I-labeled IGF-II. A: the autoradiograph of a ligand blot, which is representative of 3 independent experiments. B: quantitative analysis of ligand blots. Values are means ± SE; n = 3. Values with the same letter are not significantly different within IGFBP groups.

Exogenous IGF-II abrogated growth inhibitory effects of t10c12 CLA in Caco-2 cells. Because t10c12 CLA decreased Caco-2 cell growth and the IGF-II production, we next investigated whether inclusion of IGF-II abrogates the growth inhibitory effect of the CLA isomer. Cells were incubated with or without 2.5 µM t10c12 CLA in the absence or presence of 200 nM IGF-II. As depicted in Fig. 10, 2.5 µM t10c12 CLA decreased cell growth and exogenous IGF-II decreased the growth inhibitory effect of the CLA isomer.


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Fig. 10.   Effect of IGF-II on growth inhibition of Caco-2 cells induced by t10c12 CLA. Cells were cultured in 24-well plates and serum starved as described in the Fig. 1 legend. After serum starvation, the monolayers were incubated with or without 2.5 µM t10c12 CLA in the absence or presence of 200 nM IGF-II. Cell numbers were estimated by the MTT assay. Values are means ± SE; n = 6. Values with different letters are significantly different (P < 0.05). Values with the same letter are not significantly different.

t10c12 CLA induced apoptosis of Caco-2 cells and IGF-II diminished apoptotic effect of t10c12 CLA. One of the mechanisms responsible for the growth inhibition of t10c12 CLA in Caco-2 cells could be apoptosis. To test this hypothesis, we performed the DNA fragmentation assay, which is considered a hallmark of apoptosis. As shown in Fig. 11, Caco-2 cells treated with t10c12 CLA produced a distinct oligosomal ladder with different sizes of DNA fragments, a typical characteristic of cells undergoing apoptosis. The inclusion of 200 nM IGF-II in the incubation medium decreased the DNA laddering induced by t10c12 CLA. To quantify early apoptotic cells, fluorescence-activated cell sorting analysis was performed using annexin V-PE together with 7-AAD. As shown in Fig. 12, the t10c12 isomer decreased the percentage of living cells compared with controls, and the addition of IGF-II in the incubation medium abrogated the decrease. The t10c12 isomer significantly increased apoptosis compared with controls, and IGF-II mitigated the apoptotic effect of the t10c12 isomer.


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Fig. 11.   Oligonucleosomal laddering analysis. Cells were plated and serum starved as described in the Fig. 1 legend. After serum starvation, the monolayers were incubated in the absence (Con) or presence of t10c12 CLA or t10c12 CLA + 200 nM IGF-II. DNA samples were prepared and analyzed by agarose gel electrophoresis. The photograph of the ethidium bromide-stained gel shown is representative of 3 independent experiments.



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Fig. 12.   Fluorescence-activated cell sorting. Cells were treated as described in the Fig. 11 legend, trypsinized, loaded with 7-aminoactinomycin D (7-AAD) and phycoerythrin-conjugated annexin V (annexin V-P), and then analyzed by flow cytometry. A: the cross wires were drawn so that the lower left quadrants (LL) contain the viable cells that exclude 7-AAD and are negative for annexin V. The upper right quadrants (UR) contain the late apoptotic cells, which are positive for both 7-AAD and annexin V. The lower right quadrants (LR) show the early apoptotic cells that are positive for annexin V but negative for 7-AAD. Results shown are representative of 6 independent experiments. UL, upper left quadrants. B: the number of living cells and early apoptotic cells is expressed as the % of total cell number. Values are means ± SE from 6 independent experiments. Values with different letters are significantly different (P < 0.05). Values with the same letter are not significantly different.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We (33) and Liew et al. (24) have previously reported that CLA inhibited colon tumorigenesis of animals injected with chemical carcinogens. Earlier in vitro studies (30, 40) also provided results showing that CLA inhibited colon cancer cell growth. As the commercial CLA preparations used in these earlier studies contained predominantly the t10c12 and c9t11 isomers (31), we examined which isomer is the more potent inhibitor of cell growth. The present study provides the first evidence that the t10c12 isomer, not the c9t11 isomer, of CLA inhibits colon cancer cell growth.

Cancer is frequently described as a disorder of the balance between cell proliferation and cell death. We (33) have previously observed that dietary CLA increased the apoptotic index in the colonic mucosa of rats injected with dimethylhydrazine (DMH), indicating that CLA inhibits DMH-induced colon carcinogenesis by inducing apoptosis. Ip and co-workers (18) also reported that CLA induced apoptosis and reduced the expression of bcl-2 in premalignant lesions of the rat mammary gland but not in normal mammary gland. The present study provides direct evidence that t10c12 CLA induces apoptosis of Caco-2 cells, indicating that dietary CLA inhibits colon cancer cell growth by inducing apoptosis. However, [3H]thymidine-incorporation studies showed that the inhibition of Caco-2 cell growth by t10c12 CLA was also due, at least in part, to decreased cellular proliferation. The effect of CLA on the cell cycle was studied by Durgam and Fernandes (11) using MCF-7 human breast cancer cells. Their results (11) indicated that the number of cells in the G0/G1 phase was higher in cells treated with CLA compared with cells treated with LA or control cells.

As shown in Fig. 2, the effect of the CLA mixture on viable cell number was much smaller than that of t10c12 CLA. The mixture used in the present study contained all possible isomers of CLA (31). Our previous results (unpublished findings, E. J. Kim and J. H. Y. Park) showed that the mixture produced similar dose-dependent growth-inhibiting effects at 10-fold higher concentrations than those of t10c12 CLA utilized in the present study. It can be speculated that CLA isomer(s), other than t10c12 and c9t11, presented in minor amounts in the mixture may have growth-stimulating effects since c9t11 CLA did not change viable cell numbers.

To understand the mechanisms by which CLA decreases colon cancer cell growth, the current study examined the influence of CLA isomers on the secretion of IGF-II and IGFBPs by Caco-2 cells. Caco-2 cells express IGF-II mRNA, but IGF-I mRNA expression was not detected (34). Zarrilli et al. (49) have shown that the levels of IGF-I receptors and IGF-II secretion are high in proliferating cells and reduced in differentiated cells. Monoclonal antibodies against the IGF-I receptor inhibit basal and IGF-II-stimulated cell proliferation, and the sustained expression of IGF-II increases proliferative rate (50). This indicates that IGF-II is an autocrine growth factor for Caco-2 cells. It has also been reported (36) that the mitogenic potency of pro-IGF-II was greater than that of mature IGF-II for human fetal-derived fibroblasts, and both IGF-II forms were more potent than IGF-I. In the present study, the t10c12 isomer decreased cell numbers and IGF-II secretion in Caco-2 cells. The decrease in pro-IGF-II was greater than that of mature IGF-II. In addition, exogenous IGF-II abrogated the growth-inhibiting effect of the t10c12 isomer. Furthermore, exogenous IGF-II decreased apoptosis induced by the CLA isomer. These results indicate that decreased concentrations of IGF-II may be, at least in part, responsible for the apoptosis and reduced DNA synthesis in cells treated with the t10c12 isomer.

The present data indicate that the decrease in IGF-II production by t10c12 CLA was due, at least partly, to the decrease in mRNA levels. However, the correlation between IGF-II protein and mRNA levels after CLA treatments was not high. The lack of correlation between IGF-II protein and mRNA can be easily explained by the characteristics of the IGF-II gene. As reported previously (41), Caco-2 cells express both P3- and P4-derived transcripts, giving rise to 6- and 4.8-kb mRNA, respectively (Figs. 6 and 7). P3 transcripts contain the P3 leader, which is 1,171 nt long. The sequence is very CG rich, so the leader contains many secondary structures that make translation difficult. P4 transcripts contain the P4 leader, which is only 109 nt long and contains no significant secondary structures. In fact, De Moor et al. (8) showed that the different IGF-II leaders significantly affect the translation efficiency of the various IGF-II messengers. Transcripts with leader 4 (from P4) are efficiently translated, whereas leader 3 (from P3) has a strong repressive effect on translation so P3 transcripts do not translate very well. In addition, the site-specific endonucleolytic cleavage in the 3'-UTR of IGF-II mRNAs occurs for both P3 and P4 transcripts and there may be differences in translation efficiency between P3 and P4 transcripts due to this endonucleolytic cleavage and the stability of the messengers. This effect is present but it is not strong. We (47) have previously demonstrated that cleavage of IGF-II mRNAs occurs more often in P3 transcripts (140%) than in P4 transcripts (100%), but this difference is not very large. Furthermore, Singh et al. (41) have shown that both the P3 and P4 promoters are transcriptionally active in Caco-2 cells and that their activity may play a selective role in regulating IGF-II mRNA levels during growth and differentiation. Using the sensitive method of RNA protection assays, Singh et al. (41) have shown that P4-derived IGF-II transcripts play a significant role in Caco-2 cells.

The above-mentioned characteristics of the IGF-II gene explain why the correlation between IGF-II protein and mRNA levels after CLA treatments was not high. In Fig. 4, it is shown that t10c12 treatment decreased the amount of IGF-II protein. In Fig. 6, it is shown that the amounts of P3 6-kb mRNA were minimally changed, whereas the amounts of P4 4.8-kb mRNA were significantly reduced at increasing t10c12 concentrations (Fig. 5). This makes sense because the P4 transcripts contribute most to IGF-II translation (8) so the decrease in IGF-II protein levels would be larger than the decrease in mRNA levels. The fact that P3 transcripts were not as downregulated is not that important because in this case P3 transcripts did not contribute much to the IGF-II protein content. In addition, it is shown in Fig. 5 that c9t11 treatment did not increase IGF-II protein content. However, it is clearly visible that the amount of the P3 transcripts of 6 kb increased when cells were incubated with higher concentrations of c9t11 (Fig. 7), whereas the P4 transcripts were not changed significantly. Because translation of the P3 transcripts did not contribute that much to IGF-II protein synthesis, the total amount of IGF-II protein (Fig. 5) did not change.

The mechanisms by which the t10c12 isomer regulates IGF-II gene regulation were not explored in the present study. CLA may compete with LA for the delta-6 desaturase, elongase, and the delta-5 desaturase pathway and thereby alter arachidonic acid (AA) and membrane phospholipid synthesis (25). In addition to decreased AA levels, polyunsaturated fatty acids of 18 or 20 carbons with a conjugated diene bond are reported (4, 28, 29) to be powerful inhibitors of the cyclooxygenase and lipoxygenase enzymes. It has also been reported (4) that CLA does not serve as a substrate for cyclooxygenase. Therefore, it was expected that the inclusion of CLA in culture medium would decrease eicosanoid synthesis. We have previously observed that PGE2 levels in the colonic mucosa were significantly decreased in rats fed a diet containing 1% CLA (33). In addition, Liu and Belury have shown that CLA reduces AA content and PGE2 synthesis in murine keratinocytes (25). In osteoblasts, PGE2 has been shown (46) to activate IGF-I gene expression through a cAMP-dependent protein kinase A pathway. Furthermore, Ricchi et al. (39) have shown that aspirin, a powerful cyclooxygenase inhibitor, reduces Caco-2 cell replication and IGF-II mRNA expression. These results suggest that aspirin and CLA inhibit IGF-II gene expression by the same mechanisms involving inhibition of prostaglandin synthesis. However, we observed in the present study that the c9t11 isomer seems to exert an opposite effect on IGF-II gene expression. It is still possible that both the t10c12 isomer and aspirin induce G0/G1 cell cycle arrest, and the reduced IGF-II expression may be simply related to the cell cycle arrest induced by these compounds.

Alternatively or as a part of coordinated response, t10c12 CLA or its metabolites may activate the peroxisomal proliferator-activated receptor (PPAR) transcription factor family. Houseknecht et al. (14) have shown that mRNA levels of aP2, a PPAR-responsive gene in the adipose tissue of fatty Zucker diabetic rats, were increased as a result of PPAR gamma activation by CLA. The two promoters of the IGF-II gene (P3 and P4) actively expressing IGF-II in Caco-2 cells contain several putative peroxisome proliferator response elements. One could speculate that the P4 promoter would be the better candidate for regulation by PPAR since we observed the stronger regulatory effect of t10c12 CLA on this promoter. It remains to be determined whether t10c12 CLA inhibits IGF-II gene expression by activating PPAR and/or decreasing PGE2 synthesis. Conversely, IGF-II was reported (10) to upregulate cyclooxygenase-2 and PGE2 synthesis in Caco-2 cells.

Caco-2 cells secrete IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6, and secretion of these binding proteins changes during the course of cell growth and differentiation (34, 52). Under the conditions of the present experiment, IGFBP-2, IGFBP-4, and IGFBP-6 were the predominant forms of IGFBP secreted by Caco-2 cells. In the current study, although the three IGFBPs were significantly (P < 0.01) decreased by the t10c12 isomer, the changes in IGFBP-4 and IGFBP-6 secreted by Caco-2 cells were not substantial compared with controls. The secretion of IGFBP-2 was somewhat decreased. Among their diverse physiological activities, IGFBPs are able to negatively modulate the actions of IGFs by binding to IGFs and preventing them from binding to IGF-I (1). However, in human colon carcinoma, IGFBP-2 mRNA (27) was reported to be overexpressed compared with normal adjacent tissues, and circulating IGFBP-2 was elevated (38). In addition, preliminary studies by Miraki-Moud et al. (26) have shown that IGFBP-2-overexpressing Caco-2 cells grew at a faster rate compared with controls, indicating that IGFBP-2 stimulates cell proliferation. These results support our hypothesis that downregulation of IGFBP-2 by CLA may lead to less cell growth.

In conclusion, the present study provides the first evidence that the growth inhibitory effect of CLA on Caco-2 colon cancer cells is attributed to the action of the t10c12 isomer. The t10c12 isomer decreased the secretion of Mr 7,500 mature and Mr 11,000-14,300 pro-IGF-II in a concentration-dependent manner, and exogenous IGF-II abrogated the growth inhibitory effect of the t10c12 isomer. These results suggest that the decrease in Caco-2 cell number by CLA may, at least in part, be mediated by decreasing IGF-II secretion that is an autocrine growth stimulator of Caco-2 cells. Future studies are required for a more complete understanding of the specific mechanisms for the effect of the t10c12 isomer on cancer cell proliferation.


    ACKNOWLEDGEMENTS

We thank M. H. Niedenthal of Lilly Research Laboratories for recombinant IGF-II.


    FOOTNOTES

This research was supported by Hallym Academy of Sciences at Hallym University Grant 2002-1 and Korea Science and Engineering Foundation Grant R01-1999-00166.

A portion of this work was presented previously at the 83rd Annual Meeting of the Endocrine Society in Denver, CO, in June 2001.

Address for reprint requests and other correspondence: J. H. Y. Park, Division of Life Sciences, Hallym Univ., 1 Okchon Dong, Chunchon, 200-702, Korea (E-mail: jyoon{at}hallym.ac.kr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 3, 2002;10.1152/ajpgi.00495.2001

Received 19 November 2001; accepted in final form 26 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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