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Conjugated linoleic acid downregulates insulin-like growth factor-I

Posted on: Thursday, 28 August 2003, 06:00 CDT

Manuscript received 14 March 2003. Initial review completed 1 April 2003. Revision accepted 29 April 2003.

ABSTRACT Conjugated linoleic acid (CLA) has chemoprotective properties in a variety of experimental cancer models. We have previously observed that dietary CLA inhibits colon tumorigenesis induced by 1,2-dimethylhydrazine in rats. In addition, our in vitro studies have shown that CLA inhibits DNA synthesis and induces apoptosis in HT-29 cells, the human colon adenocarcinoma cell line. The insulin-like growth factor (IGF) system regulates the growth of HT-29 cells by an autocrine mechanism. The present study examined whether the growth inhibitory effect of CLA is related to changes in the IGF system in HT-29 cells. To determine whether CLA inhibits IGF- II production, HT-29 cells were incubated in serum-free medium in the presence of various concentrations of CLA. CLA decreased protein levels of both mature and pro IGF-II and IGF-II transcripts. Whereas exogenous IGF-I and IGF-II produced an increase in cell number, neither IGF-I nor IGF-II counteracted the negative growth regulatory effect of CLA. Reverse transcriptase-polymerase chain reaction and Western blot analysis of total cell lysates revealed that CLA decreased IGF-I receptor (IGF-IR) transcript and protein levels in a dose-dependent manner. Immunoprecipitation/Western blot studies revealed that CLA inhibited IGF-I-induced phosphorylation of IGF-IR and insulin-receptor substrate (IRS)-I, recruitment of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) to IGF- IR, IGF-IR-associated PI3K activity, and phosphorylated Akt and extracellular signal-regulated kinase (ERK)-1/2 levels. In conclusion, the inhibition of cell proliferation and induction of apoptosis by CLA in HT-29 cells may be mediated in part by its ability to decrease IGF-II synthesis and to downregulate IGF-IR signaling and the PI3K/Akt and ERK-1/2 pathways. J. Nutr. 133: 2675- 2681, 2003.

KEYWORDS: [middot] insulin-receptor substrate-1 [middot] Akt [middot] extracellular signal-regulated kinase

Conjugated linoleic acid (CLA)3 is the common element of a group of C18 fatty acids with two double bonds exhibiting strong anticarcinogenic effects in a variety of animal models (1). CLA is found naturally in food such as milk fat and the meat of ruminant animals. In spite of the abundance of studies reporting the anticarcinogenic properties of CLA, the molecular mechanisms that facilitate these effects are not currently well known.

The insulin-like growth factor (IGF) system consists of the peptide growth factors IGF-I and IGF-II, the type I and II IGF receptors, the IGF-binding proteins (IGFBP), and their corresponding proteases (2). Initially identified as potent physiological mitogens, IGF are now known to be polypeptides with effects on cell proliferation, differentiation, apoptosis and transformation (3). The actions of IGF are mediated by the IGF-I receptor (IGF-IR). Similar to the insulin receptor in structure, the IGF-IR is a heterotetrameric glycoprotein with two extracellular [alpha]- subunits and two transmembrane [beta]-subunits. Ligand binding to the receptor induces receptor autophosphorylation in the intracellular domain of the [beta];-subunit and results in activation of the intrinsic tyrosine kinase of the IGF-IR. Signaling pathways known to be activated by IGF-IR include the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) (4).

Insulin-receptor substrate (IRS)-1, IRS-2, and She are immediate substrates of the IGF-IR and are phosphorylated after binding to the activated receptor through its phosphotyrosine-binding domain. IRS- 1 is phosphorylated on multiple tyrosine residues that serve as docking sites for a variety of signaling molecules including the p85 regulatory subunit of PI3K (4). Tyrosine-phosphorylated IRS-1 binds p85 and thereby activates the associated catalytic subunit (p110) of the enzyme (5). The serine/threonine kinase Akt, or protein kinase B (PKB) appears to be critical for a variety of cellular signaling pathways and serves as a transducer of multiple functions initiated by growth factor receptors that activate PI3K. Tyrosine phosphorylated Shc activates the Ras-ERK signaling pathway through a Grb2-Son of Sevenless complex (6). PI3K, through its downstream target Akt/PKB, as well as the MAPK pathway, promotes growth factor- mediated mitogenesis and blocks programmed cell death or apoptosis (7,8).

The aberrant activation of the IGF-IR induces growth, neoplastic transformation, and tumorigenesis (9). The critical role played by the IGF-IR in the development of tumors suggests that this receptor might be an attractive target for dietary intervention for cancer prevention. Colon cancer is one of the most frequent malignant diseases in the developed world, and experimental and clinical data implicate the IGF-IR in colon cancer etiology. Compared with normal tissues, the IGF-IR is overexpressed by tumors in colorectal cancer (10,11). In addition, IGF-I protects colon cancer cells from death factor-induced apoptosis (12). Furthermore, IGF-II mRNA is overexpressed in human colon carcinoma compared with normal adjacent tissues (13). Therefore, the discovery of agents that inhibit the IGF-I signaling pathway could lead to the development of highly successful prevention strategies for colon cancer.

Dietary CLA inhibits colon tumor incidence in rats treated with 1,2-dimethylhydrazine (14). In vitro studies have shown that CLA inhibits cell proliferation and induces apoptosis of HT-29 cells, the human colon adenocarcinoma cell line (15). The present study examined whether the growth inhibitory effect of CLA is related to changes in the IGF system in HT-29 cells. HT-29 cells synthesize and secrete IGF-II, IGFBP-2, -4, and -6 (16), and IGF-II acts as an autocrine growth regulator of these cells (17). Therefore, this study examined the effect of CLA on IGF-II production and the activation of several key proteins in the IGF-I signal transduction pathway.

MATERIALS AND METHODS

Reagents. Reagents were purchased from suppliers as follows: monoclonal anti-[beta]-actin, essentially fatty acid-free bovine serum albumin (BSA), a mixture of CLA isomers (18), and 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO); DMEM/Ham's F-12 nutrient mixture (DMEM/ F12), fetal bovine serum (FBS), transferrin, and selenium (Life Technologies, Gaithersburg, MD); horseradish peroxidase (HRP)- conjugated anti-rabbit and anti-mouse Ig (Amersham, Piscataway, NJ); anti-PI3K p85 and anti-IRS-1 antibody (Upstate Biotechnogy, Lake Placid, NY); anti-Akt (29752), anti-phospho-Akt (p-Akt, 473) and [[gamma]-^sup 32^P]ATP (NEN Life Sciences, Boston, MA); anti- phosphotyrosine-RC20 antibody linked to HRP (PY20; BD Transduction Laboratories, Palo Alto, CA); antibodies against phospho-p44/42 MAP kinase (p-ERK-1/2, Thr202/Tyr203), p44/42 MAP kinase (ERK-1/2), phosphoinositide-dependent protein kinase 1 (PDK-1), p-PDK-1, phosphatase and tensin homologue deleted on chromosome ten (PTEN), and p-PTEN (Cell Signaling Technology, Beverly, MA); and antibodies against IGF-IR[beta]; (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA). Recombinant human IGF-I and IGF-II were generously provided by Genentech (San Francisco, CA) and Eli Lilly (Greenfield, IN), respectively.

Cell culture. The HT-29 cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM/F12 containing 100 mL/L of FBS with 100,000 U/L penicillin and 100 mg/L of streptomycin. HT-29 cells between passages 135 and 145 were used in these studies. To examine the effect of CLA and IGF, cells were plated in 24-well plates at 50,000 cells/well with DMEM/F12 containing 100 mL/L of FBS. Prior to CLA treatment, the cell monolayers were rinsed and serum starved for 24 h with DMEM/F12 supplemented with 5 mg/L of transferrin, 0.1 g/L of BSA and 5 [mu]g/ L of selenium (serum-free medium). After serum starvation, fresh serum-free medium containing the indicated concentrations of CLA and/ or recombinant human IGF-I or IGF-II was replaced. Fatty acids were complexed to essentially fatty acid-free BSA, with the molar ratio of fatty acid to BSA being 4:1 (19). Media were changed every two days. Viable cell numbers were estimated by the MTT assay as described previously (20).

IGF-II immunoblot analysis. HT-29 cells were cultured as described above. Conditioned media (24-h) were collected between, d 2 and 3 of culture, concentrated 10-fold and used for immunoblot analysis of IGF-II as described previously (20). The relative abundance of each band was measured by a densitometric scanning of the exposed films using the Bio-profile Bio-1D application (Vilber- Lourmat, France).

Immunoprecipitation and immunoblotting analyses. Cell lysates were prepared as previously described (15). For immunoprecipitation, cell lysates (0.75 mg protein) were precleared by incubation on a rotating platform for 1 h at 4[degrees]C with 1 [mu]g of normal rabbit IgG and 50 [mu]L of a resuspended volume of protein A- Sepharose beads (Amersham) and centrifuged at 1000 x g for 5 min at 4[degrees]C. The supernatants were incubated with 1 [mu]g of anti- IGF-IR[degrees] antibody for 2 h at 4[degrees]C. Protein A- Sepharose beads were added to the lysate-antibody mix, which was then incubated for 2 h at 4[degrees]C. The beads were washed four times with lysis buffer. The immunoprecipitates or total cell lysates were resolved on sodium dodecyl sulfate (SDS), 40-200 g/L of polyacrylamide gel and transferred onto polyvinylidene fluoride membrane (Millipore). The blots were blocked for 1 h in 10 g/L of BSA in TBS-T (20 mmol/L Tris-Cl, pH 7.5, 150 mmol/L NaCl, 1 g/L Twcen 20) or 50 g/L of milk TBS-T and incubated for 1 h with either anti-phospho-Tyr (PY20-HRP, 1:5,000), anti-IGF-IR[beta] (1:500), anti-IRS-1 (1:720), anti-PDK (1:1000), anti-Akt (1:1000), anti-p- Akt (1:1000), anti-ERK-1/2 (1:1000), anti-p-ERK-1/2 (1:1000), anti- PDK-1 (1:1000), anti-p-PDK-1 (1:1000), anti-PTEN (1:1000), anti-p- PTEN (1:1000), or anti-[beta]-actin (1:2000) antibody. The blots were then incubated with anti-mouse or anti-rabbit HRP-conjugated antibody. Signals were detected by using the enhanced chemiluminescence method using SuperSignal west dura extended duration substrate (Pierce, Rockford, IL). The relative abundance of each protein band was analyzed by densitometric scanning of the exposed films. Immunoblots were probed with an antibody for [beta]- actin as a control for protein loading.

Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated using the guanidium isothiocyanate- phenolchloroform method and RT-PCR was performed as previously described (21). Each PCR cycle consisted of denaturing at 94[degrees]C for 1 min, annealing at temperatures listed in Table 1 for 1 min and extending at 72[degrees]C for 1 min. Sequences for PCR primer sets and numbers of cycles used for PCR amplication are listed in Table 1. The relative abundance of each band was estimated by densitometric scanning of the exposed films. The levels of mRNA were corrected as a ratio to the corresponding [beta]-actin level.

PI3K assay. An assay for PDK activity was performed as previously described (22). Cell lysate (0.75 mg protein) was immunoprecipitated with polyclonal antibody against IGF-IR[beta] followed by incubation with protein A-Sepharose beads. After washing, the beads were resuspended in 20 [mu]L of kinase buffer (20 mmol/L Hepes, pH 7.2, 50 mmol/L NaCl, 1 mmol/L EGTA) containing 4 [mu]g of phosphatidylinositol (Sigma), 10 [mu]mol/L of ATP, 5 mmol/L of MnCl^sub 2^, and 10 [mu]Ci of [[gamma]-^sup 32^P]ATP and incubated for 20 min at 30[degrees]C. The resulting ^sup 32^P-labeled phosphatidylinositol 3-phosphate (PIP) lipids were separated from other reaction products by TLC and visualized by autoradiography. The radioactive PIP signals were quantitated by densitometry.

Statistical analyses. For all studies, 3-6 independent experiments were performed with separate batches of cells. For each independent experiment duplicate samples were analyzed. Data were analyzed by one-way, two-way, or two-factor repeated measurements ANOVA and are expressed as the mean + or - SEM. Differences between treatment groups were analyzed by Duncan's multiple range test or t test. Means were considered significantly different at P < 0.05. All statistical analyses were done using the SAS System for Windows V8 (SAS Institute, Cary, NC).

TABLE 1

Primer sequences used for PCR amplification1

RESULTS

CLA decreases IGF-II transcript and protein expression. To examine the effect of CLA on IGF-II production of HT-29 cells, monolayer cultures were treated with CLA (0-20 [mu]mol/L) in serum- free medium, and the IGF-II level in conditioned media was determined by Western blot analysis. CLA decreased the levels of both pro (M^sub r^ 14,300) and mature (M^sub r^ 7500) IGF-II in a dose-dependent manner (Fig. 1A). Results of the RT-PCR analysis revealed that CLA decreased IGF-II transcripts in HT-29 cells in a dose-dependent manner (Fig. 1B).

To determine whether exogenous IGF-I or IGF-II would counteract growth inhibition induced by CLA, serum-starved cells were cultured with or without 10 nmol/L of IGF-I or IGF-II in the absence or presence of 20 [mu]mol/L of CLA for 2 or 4 d. Cells cultured in the presence of CLA grew at a slower rate, and both IGF-I and IGF-II mitigated the growth inhibitory effect of CLA at d 2 (Fig. 2). Both IGF-I and IGF-II alone also increased viable cell number at both d 2 and 4. However, at d 4, the viable cell number was not higher in either CLA + IGF-I-treated or CLA + IGF-II-treated group compared with the CLA-treated group. Thus, neither IGF-I nor IGF-II stimulation of IGF-IR signaling was sufficient to counteract the negative influence of CLA on HT-29 cell growth, suggesting that CLA may downregulate IGF-IR signaling.

CLA downregulates IGF-I receptor levels. To investigate whether CLA influences IGF-IR expression in HT-29 cells, total cell lysates were immunoblotted with an antibody specific for IGF-IR[beta]. Two bands with M^sub r^ 95,000 (mature IGF-IR [beta]-subunit) and 200,000 (IGF-IR precursor) were detected. Treatment of HT-29 cells with increasing concentrations of CLA led to decreased mature IGF- IR levels but increased IGF-IR precursors (Fig. 3A). To determine whether CLA regulates the expression of IGF-IR at a transcriptional level, mRNA levels were determined by RT-PCR analysis. CLA decreased IGF-IR transcripts (Fig. 3B).

To determine whether CLA affects IGF-IR tyrosine phosphorylation, cells were cultured in the absence or presence of 20 [mu]mol/L of CLA for 3 d and lysates were prepared after 0, 1, 5, or 60 min of stimulation with 10 nmol/L of IGF-I. Levels of IGF-IR expression and phosphorylation were measured by immunoprecipitation followed by immunoblotting. After addition of IGF-I, IGF-IR levels remained constant and IGF-IR phosphorylation increased 15-fold within 1 min with a decrease at 5 min, indicating that the receptor was being activated by the ligand (Fig. 4). IGF-IR protein levels were decreased by 25% in cells treated with 20 [mu]mol/L of CLA (P < 0.05). IGF-I similarly induced IGF-IR activation but to a lesser degree in cells treated with CLA (P < 0.05) indicating the ability to respond to the stimulation of IGF-I. Thus CLA appeared to negatively regulate IGF-IR phosphorylation by causing a decrease in the expression levels of this signaling protein.

FIGURE 1 Effect of conjugated linoleic acid (CLA) on insulin- like growth factor (IGF)-II secretion and transcripts in HT-29 cells. Cells were cultured in serum-free medium in the presence of 0- 20 [mu]mol/L CLA. (A) IGF-II immunoblot analysis. Twenty-four-hour conditioned media were concentrated for immunoblot analysis with a monoclonal antibody against IGF-II. The relative abundance of the pro or mature IGF-II band was estimated by densitometric analysis. The volumes of media loaded onto the gel were adjusted for equivalent cell numbers. (B) IGF-II mRNA. Total RNA was isolated for RT-PCR and relative abundance of IGF-II transcript band was estimated by densitometric analysis. Each bar represents the mean + or - SEM (n = 3). Means without a common letter differ, P < 0.05.

FIGURE 2 Effect of conjugated linoleic acid (CLA) and/or insulin- like growth factors (IGF) on viable HT-29 cell number. Cells were cultured in serum-free medium in the absence or presence of 20 [mu]mol/L CLA and/or 10 nmol/L IGF-I or IGF-II. Viable cell numbers were estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide assay. Each bar represents the mean + or - SEM (n = 6). Means without a common letter differ, P < 0.05.

Western blot analysis of total lysate revealed that CLA did not influence the levels of IRS-1 protein (data not shown). Lysates from IGF-I-treated cells were immunoprecipitated with anti-IGF-IR antibody, and the immunoprecipitates were immunoblotted with an antibody directed to phosphotyrosine or IRS-1. Anti-IGF-IR[beta] antibody coimmunoprecipitated IRS-1 in a time-dependent manner that was consistent with tyrosine phosphorylation of IRS-1 (Fig. 4). Tyrosine phosphorylated IRS-1 was maximal at 1 min and decreased thereafter. CLA decreased both phosphorylation of IRS-1 and its association to IGF-IR.

IGF-I-induced activation of PI3K and Akt is inhibited by CLA. Expression of the p85 regulatory subunit of PI3K in HT-29 cells treated with increasing amounts of CLA was examined by the use of immunoblotting. Whereas decreased levels of mature IGF-IR were detected (Fig. 3A), expression of PI3K was not altered by addition of CLA (data not shown). CLA influences on the IGF-I-induced association of IGF-IR and PI3K were determined by the use of immunoprecipitation with an anti-IGF-IR antibody followed by immunoblotting with the p85 antibody. IGF-I stimulated the association of the p85 regulatory subunit of PI3K with IGF-IR, and the association was reduced in CLA-treated cells (Table 2). The levels of PI3K associated with IGF-IR were normalized to that of IGF- IR expression to determine whether the reduced association after CLA treatment was a result of decreased IGF-IR levels or of an inhibition of p85 recruitment. After normalization and addition of CLA, the reduced association was not evident (Table 2) indicating that the decrease in PI3K association to IGF-IR was a result of decreased IGF-IR levels but not of an inhibition of p85 recruitment.

PI3K activity in the anti-IGF-IR immunoprecipitates was analyzed by in vitro kinase assays in which the product was resolved by TLC showing the spots of 32P-labeled PIP (Fig. 5). In accordance with p85 immunoblotting analysis, HT-29 cells incubated with IGF-I showed time-dependent changes in the enzymatic activity of their anti-IGF- IR immunoprecipitates. The PI3K activities increased 25-fold at 1 min after the IGF-I treatment and decreased thereafter. The PI3K activities were lower in cells treated with 20 [mu]mol/L of CLA during the 60-min IGF-I treatment.

To determine whetherIGF-I receptor activation of PI3K leads to strong activation of Akt, total cell lysates were analyzed by immunoblotting with anti-p-Akt and whole Akt protein antibodies showing the time-dependent increase in Akt activation (p-Akt) by IGF- I (Fig. 6). Barely detectable in cells grown in serum-free medium, IGF-I induced Akt activation within 5 min. The increase in p-Akt after IGF-I treatment was slightly delayed compared with the IGF-IR and PI3K activation due to the downstream position of Akt. No changes were observed in Akt protein levels during the 60-min IGF-I stimulation. However, the level of active Akt was substantially decreased in cells treated with CLA, whereas the total Akt protein levels were moderately decreased by CLA treatment (P < 0.05). Protein levels of PTEN, p-PTEN, PDK-1, and p-PDK-1 were not affected by CLA (data not shown).

IGF-I-induced activation of ERK is inhibited by CLA. To examine the effect of CLA on the ERK subfamily of MAPK, Western blotting was performed with the total lysates prepared from cells treated with or without 20 [mu]mol/L of CLA and/or 10 nmol/L of IGF-I as described above using antibodies specific for ERK-1/2 and p-ERK-1/2. IGF-I activation of IGF-IR signaling resulted in a time-dependent increase in ERK phosphorylation in HT-29 cells. Phosphorylated ERK levels were increased at 5 min after IGF-I stimulation but the magnitude of the increase was much smaller compared with that of p-Akt. Both basal and IGF-I-induced p-ERK levels were markedly decreased by 20 [mu]mol/L of CLA. Total ERK levels also decreased after CLA treatment (Fig. 7).

FIGURE 3 Effect of conjugated linoleic acid (CLA) on insulin- like growth factor (IGF)-I receptor (IGF-IR) proteins and transcripts in HT-29 cells. (A) IGF-IR immunoblot analyses. (B) IGF- IR mRNAs. Each bar represents the mean + or - SEM (n = 3). Means without a common letter differ, P < 0.05.

FIGURE 4 Effect of conjugated linoleic acid (CLA) on insulin- like growth factor (IGF)-I-induced tyrosine phosphorylation of IGF- I receptor (IGF-IR) and insulin-receptor substrate (IRS)-1 in HT-29 cells. Cells were treated in the absence or presence of 20 [mu]mol/ L CLA for three days and lysed without stimulation (0) or after 1, 5, or 60 min of stimulation with 10 nmol/L of IGF-I. (A) Cell lysates were incubated with an anti-IGF-IR[beta] antibody and the immune complexes were precipitated with protein A-Sepharose. The immunoprecipitated proteins were analyzed by Western blotting with antibodies against phosphotyrosine (PY-20), IGF-IR[beta], or IRS-1. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. (B) Quantitative analysis of immunoblots. Each bar represents the mean + or - SEM (n = 3). *Different from - CLA at each time point, P < 0.05.

TABLE 2

Effect of CLA on IGF-I-induced recruitment of the p85 subunit of PI3K to IGF-IR in HT-29 cells1-3

DISCUSSION

We have previously shown that dietary CLA lowers colon tumor incidence in rats treated with 1,2-dimethylhydrazine (14). In vitro studies have also shown that CLA inhibited the growth of SW480 (23) and HT-29 cells (24,25), the human colon cancer cell lines. CLA inhibits HT-29 cell growth by both decreasing cell proliferation and inducing apoptosis (15). The present study examined the hypothesis that CLA decreases cell proliferation and induces apoptosis by changing the IGF system in HT-29 cells.

This study provided the first evidence that CLA reduces IGF-IR protein expression and IGF-IR-mediated signaling, which is consistent with the finding that CLA blunts the effect of exogenous IGF-I on HT-29 cell growth. When HT-29 cells were incubated with IGF- I or IGF-II in serum-free medium, the cells responded by increasing in number. However, treatment of HT-29 cells with 20 [mu]mol/L of CLA for 96 h completely inhibited the IGF-I-induced increase in cell number, suggesting that CLA attenuated the IGF-IR signaling pathway. Indeed, decreased levels of the IGF-IR were noted at 72 h after treatment with CLA. The autocrine production of IGF-IR-binding ligands, IGF by tumor cells or the tumor-induced production of ligands by surrounding stromal cells has also been implicated in IGF- IR-mediated tumor growth (10). The expression of IGF-II in HT-29 cells was also decreased by CLA, and IGF-II is an autocrine growth regulator of HT-29 cells (26). Therefore, it is likely that CLA inhibits HT-29 cell growth by limiting the production of this growth factor and the ability to respond to IGF-II. The effect of CLA was specific for the IGF system because no effect of CLA on other important proteins (e.g., IRS-1, p85, PTEN, p-PTEN, PDK-1, and p- PDK-1) in these cells was observed.

FIGURE 5 Effect of conjugated linoleic acid (CLA) and/or insulin- like growth factor (IGF)-I on IGF-I receptor (IGF-IR)-associated phosphatidylinositol 3-kinase (PI3K) activity in HT-29 cells. Cell lysates (0.75 mg protein) were immunoprecipitated with an anti-IGF- IR[beta] antibody as described in Figure 4. The immune complexes were incubated with phosphatidylinositol and [[gamma]-^sup 32^P]ATP. Phosphatidylinositol 3-phosphate (PIP) generated by immunoprecipitated PI3K was separated by TLC. (A) An autoradiograph of the TLC plate is representative of three independent experiments. (B) Relative fold changes in PIP. Each bar represents the mean + or - SEM (n = 3). *Different from -CLA at each time point, P < 0.05.

FIGURE 6 Effect of conjugated linoleic acid (CLA) on insulin- like growth factor (IGF)-I-induced Akt activation in HT-29 cells. Similar protein samples as those in Figure 4 were analyzed by immunoblotting with anti-phospho-Ser473 Akt (p-Akt) or whole Akt protein antibodies. (A) Photographs of chemiluminescent detection of the blots are representative of three independent experiments. (B) Quantitative analysis of immunoblots. The relative fold change in p- Akt to its own Akt control band on Western blots was quantitated by densitometric analysis. Each bar represents the mean + or - SEM (n = 3). *Different from -CLA at each time point, P < 0.05.

In addition to autocrine/paracrine effects, IGF has been reported to exert an effect on colon cancer by an endocrine mechanism. Long- term prospective studies have clearly shown that high circulating levels of IGF-I and low levels of IGFBP-3 are associated with a higher risk of developing colorectal cancer (27,28). A recent case- control study in Northern Sweden has also shown a positive relationship between the levels of plasma IGF-I and colon cancer risk (29). Studies utilizing liver-specific IGF-I-deficient mice have shown that circulating IGF-I levels regulate colon cancer growth and metastasis (30). CLA could decrease IGF-I production in the liver and other tissues and thereby reduce serum levels of IGF, which could be one of the mechanisms by which CLA reduces colon tumor incidence in animals. Future studies are needed to determine whether dietary CLA reduces serum IGF levels in animals.

CLA decreased IGF-II and IGF-IR mRNA levels suggesting that CLA regulated the expression of these proteins at the transcriptional level. The present study did not determine the mechanisms responsible for the CLA regulation of these transcript levels. CLA has been reported to be an activator of the peroxisome proliferator- activated receptor (PPAR)[gamma] (31). CLA or its metabolites may influence transcription of genes that regulate growth by acting as a ligand for the PPAR. Ligands for PPAR[gamma] induce apoptosis and exert antiproliferative effects on several carcinoma cell lines (32,33). The two promoters of the IGF-II gene, P3 and P4, each contains several putative peroxisome proliferator response elements. It remains to be determined whether CLA inhibits expression of IGF- II and IGF-IR transcripts by activating PPAR[gamma]. In addition to reduced IGF-IR transcripts by CLA, we observed that CLA increased pro IGF-IR levels indicating that CLA also regulates the expression of this protein at the post translational level.

Activation of the IGF-IR requires tyrosine phosphorylation of the [beta]-subunits of the receptor. CLA decreased the protein levels of the mature IGF-I receptor in HT-29 cells in a dose-dependent manner. IGF-I induced tyrosine phosphorylation of the IGF-IR in cells treated with CLA but the degree of the phosphorylation was lower in CLA-treated cells. CLA decreased IGF-IR levels and phosphorylation to a similar degree, suggesting that CLA inhibited activation of these receptors by decreasing receptor protein levels. In addition we observed that IGF-I stimulated the recruitment of PI3K to the IGF- I receptor, and CLA decreased IGF-IR-associated PI3K protein levels and PI3K activities. These decreases do not appear to be attributed to changes in either the PI3K protein expression or p85 recruitment but rather a result of the decrease in IGF-IR protein levels.

Akt is a downstream target of PI3K and the PI3K/Akt pathway has recently been recognized as one of the most important signals ensuring protection against apoptosis (34). The present data showed that CLA inhibited IGF-I-induced activation of Akt, which could have been due to decreased IGF-IR levels and the subsequent decrease in IGF-IR activation. The moderate decrease in Akt protein levels may also have contributed to the decreased p-Akt levels. In addition to p-Akt, p-ERK-1/2 levels were decreased in CLA-treated cells, a result of both decreased total ERK-1/2 levels and protein phosphorylation. These results implied that CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells by inhibiting the Akt and MAPK signaling pathways. Increased expression of the IGF-IR and downstream signaling proteins such as Akt and ERK are frequent events in cancer. Future studies are needed to study the effect of Akt and ERK on HT-29 cell growth.

FIGURE 7 Effect of conjugated linoleic acid (CLA) on insulin- like growth factor (IGF)-I-induced p44/42 mitogen-activated protein kinase (MARK) (ERK-1/ 2) activation in HT-29 cells. Similar protein samples as those in Figure 4 were analyzed by immunoblotting with anti-phospho-p44/42 MAPK (p-ERK-1/2) or whole p44/42 MAPK (ERK-1/2) antibodies. (A) Photographs of chemiluminescent detection of the blots are representative of three independent experiments. (B) Quantitative analysis of immunoblots. The relative fold change in p- ERK-1/2 to its own ERK-1/2 control band on Western blots was quantitated by densitometric analysis. Each bar represents the mean + or - SEM (n = 3). *Different from -CLA at each time point, P < 0.05.

In conclusion, we demonstrated that CLA negatively regulated levels of IGF-II and mature IGF-IR and subsequent activation of Akt and MAPK pathways in HT-29 cells. Inhibition of IGF-I receptor signaling may be one of the mechanisms by which CLA inhibits cancer cell growth. The activation of the IGF-I/IGF-IR system has recently been shown to be a critical event in the development of several murine and human tumors. The results reported herein indicate that inhibition of Akt phosphorylation may be a major mechanism by which CLA inhibits IGF-IR signaling and cell proliferation and induces apoptosis.

ACKNOWLEDGMENTS

We thank James H. Prather of Eli Lilly & Company for recombinant IGF-II and Dina Washington of Genentech, for recombinant IGF-I. We are grateful to Robert E. Lewis at Eppley Cancer Institute, University of Nebraska Medical Center for his critical review of this manuscript.

0022-3166/03 $3.00 (C) 2003 American Society for Nutritional Sciences.

1 Funding provided by grant No. [R01-1999-000-00166-0 (2002)] from the Basic Research Program of the Korea Science & Engineering Foundation, the research grant from Hallym University, Korea, and the Korean Science and Engineering through the Silver Biotechnology Research Center at Hallym University (grant number: R12-2001-047- 02004-0).

3 Abbreviations used: BSA, bovine serum albumin; CLA, conjugated linoleic acid; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HRP, horseradish peroxidase; IGF, insulin-like growth factor; IGFBP, IGF-binding proteins; IGF-IR, insulin-like growth factor-I receptor; IRS, insulin-receptor substrate; MARK, mitogen-activated protein kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide; PDK, phosphoinositide-dependent protein kinase; PIP, phosphatidylinositol 3-phosphate; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PPAR, peroxisome proliferator-activated receptor; SDS, sodium dodecyl sulfate; PTEN, phosphatase and tensin homologue deleted on chromosome ten.

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Eun Ji Kim,* II-Jun Kang,* Man Jin Cho,* Woo Kyoung Kim,** Yeong- Lae Ha[dagger] and Jung Man Yoon Park*2

*Division of Life Sciences and Silver Biotechnology Research Center, Hallym University, Chunchon, 200-702, Korea, **Department of Food Science and Nutrition, Dankook University, Seoul, 140-714, Korea, and [dagger]Division of Applied Life Sciences, Graduate School, Gyeongsang National University, Chinju, 660-901, Korea

2 To whom correspondence should be addressed. E-mail: jyoon@hallym.ac.kr.

Copyright American Institute of Nutrition Aug 2003

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