1 Division of Life Sciences and 3 Department of Pathology, Hallym University, Chunchon 200 - 702; 2 Department of Food Science and Nutrition, Dankook University, Seoul 140 - 714; 4 Department of Biological Science, Sookmyung Women's University, Seoul, Korea 140 - 742; and 5 Department of Molecular Genetics, University of Illinois College of Medicine, Chicago, Illinois 60607
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ABSTRACT |
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Conjugated linoleic acid
(CLA) has chemoprotective properties in experimental cancer models, and
in vitro studies have shown that CLA inhibits HT-29 colon cancer cell
growth. ErbB2 and ErbB3 have been implicated in the development of
colon cancer, and both proteins are expressed at high levels in the
HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by
the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell
proliferation and apoptosis and the influence of CLA on the
ErbB3 signaling pathway, HT-29 cells were cultured in the presence of
CLA and/or heregulin. CLA inhibited DNA synthesis and induced
apoptosis of HT-29 cells. Although the addition of
heregulin- led to an increase in cell number, it was not able to
counteract the negative growth regulatory effect of CLA.
Immunoprecipitation/Western blot studies revealed that CLA inhibited
heregulin-
-stimulated phosphorylation of ErbB2 and ErbB3,
recruitment of the p85 subunit of phosphoinositide 3-kinase
(PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase
activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3
mRNA and protein levels in a dose-dependent manner. In conclusion, we
demonstrate that CLA inhibits cell proliferation and stimulates
apoptosis in HT-29 cells and that this may be mediated by its
ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.
heregulin; phosphoinositide 3-kinase; apoptosis
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INTRODUCTION |
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CONJUGATED LINOLEIC ACID (CLA) is the common denomination of a group of C18 fatty acids with two double bonds consisting of a mixture of positional and geometric isomers. CLA is a naturally occurring substance in food sources, such as milk fat and the meat of ruminant animals. It exhibits chemoprotective effects in several tissues in experimental animals, such as chemically induced forestomach neoplasia, skin tumors in mice, and mammary and colon carcinogenesis in rats (4). Inhibition of mammary tumors in rats is effective, regardless of type or amount of dietary fat (19). We (37) previously reported that dietary CLA inhibited colon cancer incidence in rats treated with 1,2-dimethylhydrazine. In addition to animal studies, in vitro studies have shown that CLA inhibits the growth of the human colon cancer cells, SW480 (34) and HT-29 (35, 42). Strong evidence for the anticancer abilities of CLA indicates a need to study the mechanisms of chemoprotection by CLA.
The ErbB family of receptor tyrosine kinases includes the epidermal
growth factor receptor (EGFR) or ErbB1, -2, -3, and -4. Activation of
these receptors regulates a number of processes including cell
proliferation, survival, and differentiation. Ligands that bind to and
activate ErbB receptors belong to two classes, those that bind the
EGFR, such as EGF and transforming growth factor- (2),
and those that bind to ErbB3 and -4, the heregulins (HRGs)
(45). Although it is devoid of any kinase activity
(14), ErbB3 is an important mediator of HRG actions. HRG
binding induces ErbB3 to associate with other members of the ErbB
family to form receptor heterodimers (2).
Overexpression of ErbB genes, particularly ErbB2, has been observed in several types of human cancer (15, 41, 49). In colon cancer, the expression of mRNA for ErbB2 and -3 as well as the corresponding proteins was increased compared with normal mucosa (6, 30, 40). However, no difference in EGFR protein levels was evident between normal colon and cancer (30). In addition, HRG is coexpressed with ErbB2 proteins in human colon cancer specimens and autocrine activation of ErbB2 occurs through heterodimerization with ErbB3 in GEO colon cancer cells (48). These results suggest that regulation of the HRG/ErbB2/ErbB3 pathway may be important modulators of aberrant growth in colon cancer (21, 33).
One of the many initial events that occur after growth factors bind to their cognate growth factor receptor tyrosine kinases is the recruitment and activation of phosphoinositide 3-kinase (PI3-kinase) (47). PI3-kinase phosphorylates inositol phospholipids at position 3 of the inositol ring and PI3-kinase lipid products interact with certain proteins and modulate their localization and/or activity (46). PKB or Akt is known as an important downstream target for PI3-kinase, which can be activated by a variety of growth factors and cytokines via phosphorylation on serine and threonine residues (7, 17, 25) and may participate in growth factor-stimulated cell cycle (10, 31) and inhibition of apoptosis (27). Disturbance of normal PKB/Akt signaling has been reported in several human cancers (20, 32). In addition to the PI3-kinase/Akt pathway, MAPK, also known as extracellular signal-regulated kinases (ERKs), are protein serine/threonine kinases that play a critical role in the regulation of cell growth and differentiation (18, 29). Both pathways are known to be regulated by HRG (5, 11).
Several studies suggest that CLA may have therapeutic benefits in individuals predisposed to developing colon cancer. The present study was designed to identify mechanisms underlying CLA regulation of growth and survival of colon adenocarcinoma cells. We confirmed that CLA inhibits DNA synthesis and stimulates apoptosis in HT-29 cells and determined that CLA regulation of proliferation and survival may be regulated by modulation of ErbB3 receptor signaling.
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MATERIALS AND METHODS |
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Reagents.
The following reagents were purchased from the indicated suppliers:
DMEM/Ham's F-12 nutrient mixture (DMEM/F-12), essentially fatty
acid-free BSA, ascorbic acid, -tocopherol phosphate, a mixture of
CLA isomers (36), and
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma, St. Louis, MO); FBS, trypsin-EDTA,
penicillin-streptomycin, transferrin, and selenium (Life
Technologies, Gaithersburg, MD); [methyl-3H]thymidine
(5 Ci/mmol), horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse Ig (Amersham); anti-PI3-kinase p85 antibody
(Upstate Biotechnogy); anti-Akt (29752), anti-phospho-Akt (p-Akt,
473), and [
-32P]ATP (New England Nuclear-Life
Sciences); anti-phosphotyrosine-RC20 antibody linked to HRP
(PY20-HRP; Transduction Laboratories); recombinant human HRG-
EGF domain (R&D Systems, Minneapolis, MN); antibodies against
phospho-p44/42 MAPK (p-MAPK, Thr202/Tyr203) and
p44/42 MAPK (Cell Signaling Technology); antibodies against EGFR
(1005), Neu (C-18), ErbB-3 (C-17), HRG (C-20), and ErbB-4 (C-18) (Santa
Cruz Biotechnology, Santa Cruz, CA).
Cell culture.
The HT-29 cell line was purchased from the American Type Culture
Collection (Manassas, VA) and was maintained in DMEM/F-12 containing
10% FBS with 100 U/ml penicillin and 100 µg/ml streptomycin. HT-29
cells between passages 135 and 145 were used in these studies. To examine the effect of CLA and HRG-,
cells were plated in 24-well plates at 50,000 cells/well with DMEM/F-12
containing 10% FBS. Before CLA treatment, the cell monolayers were
rinsed and serum-starved for 24 h with DMEM/F-12 supplemented with
5 µg/ml transferrin, 1 mg/ml BSA, and 5 ng/ml selenium (serum-free
medium). After serum starvation, fresh serum-free medium containing the
indicated concentrations of CLA and/or recombinant human HRG-
was
replaced. Fatty acids were complexed to essentially fatty acid-free
BSA, with the molar ratio of fatty acid to BSA being 4:1
(24). Media were changed every 2 days. The basal
serum-free medium containing 0.15 µM linoleic acid was chosen to
eliminate the possibility of an essential fatty acid deficiency. All
cultures contained ascorbic acid (50 ng/ml) and
-tocopherol
phosphate (20 ng/ml) to protect fatty acids from peroxidation. Viable
cell numbers were estimated by the MTT assay as described previously
(24).
Immunoprecipitation and immunoblotting analyses.
Cells were grown in 100-mm culture dishes, washed briefly with
ice-cold PBS containing 1 mM sodium orthovanadate
(Na3VO3) and 1 mM PMSF, and solubulized for 40 min at 4°C with lysis buffer containing (in mM) 20 HEPES, pH 7.5, 150 NaCl, 1 EDTA, 1 EGTA, 100 NaF, 10 sodium pyrophosphate, and 1 Na3VO3, plus 1% Triton X-100. The following
protease inhibitors were used (in µg/ml): 20 aprotinin, 10 antipain,
10 leupeptin, and 80 benzamidine HCl, plus 0.2 mM PMSF. The insoluble
material was removed by centrifugation at 13,000 g for 10 min and protein content was determined by using the BCA protein assay
kit (Pierce, Rockford, IL). Supernatant (0.75 mg/ml) was precleared by
incubating on a rotating platform for 1 h at 4°C with 1 µg of
normal rabbit IgG and 50 µl of resuspended volume of protein
A-Sepharose beads (Amersham Pharmacia Biotech) and centrifuged at 2,500 rpm for 5 min at 4°C. The supernatants were incubated with 1 µg
anti-ErbB3 or anti-ErbB2 antibody for 2 h at 4°C. Protein
A-Sepharose beads were added to lysate-antibody mix followed by
incubation for 2 h at 4°C. The beads were washed four times with
lysis buffer. The immunoprecipitate or total cell lysates were resolved
on a SDS-PAGE (4-20%) and transferred onto polyvinylidene
fluoride membrane (Millipore). The blots were blocked for 1 h in
1% BSA in 20 mM Tris · HCl, pH 7.5, 150 mM NaCl,
0.1% Tween 20 (TBST) or 5% milk TBST and incubated for 1 h with
either PY20-HRP (1:5,000), anti-EGFR (1:250), anti-ErbB2 (1:500),
anti-ErbB3 (1:1,000), anti-ErbB4 (1:500), anti-PI3-kinase (1:1,000),
anti-Akt (1:1,000), anti-p-Akt (1:1,000), anti-MAPK (1:1,000),
anti-p-MAPK (1:1,000), anti-HRG (1:500), or anti--actin
(1:2,000) 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, IL).
RT-PCR.
Total RNA was isolated by using the guanidium
isothiocyanate-phenol-chloroform method and RT-PCR was performed as
previously described (28). Each PCR cycle consisted of
denaturing at 94°C for 1 min, annealing at temperatures listed in
Table 1 for 1 min, and extension at
72°C for 1 min. Sequences for PCR primer sets and numbers of cycles
used for PCR amplification are listed in Table 1. The levels of mRNA
were corrected as a ratio to the corresponding -actin level.
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DNA laddering. HT-29 cells were cultured and treated as described above and 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 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 (30 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 various concentrations of CLA. After 3 days, cells were trypsinized and incubated with phycoerythrin-conjugated Annexin V and 7- amino-actinomycin D (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). The data were analyzed by using ModFit version 1.2 software.
PI3-kinase assay.
An assay for PI3-kinase activity was performed as previously described
(13). Cell lysate (750 µg) was immunoprecipitated with
polyclonal antibody against ErbB3 followed by incubation with protein
A-Sepharose beads. The immunoprecipitates were washed twice with 1%
Nonident P-40-PBS, twice with 100 mM Tris · HCl (pH 7.5) containing 500 mM LiCl and 1 mM
Na3VO3, and twice with 50 mM
Tris · HCl (pH 7.2) containing 150 mM NaCl. After
the last wash, the beads were resuspended in 20 µl kinase buffer (in
mM: 20 HEPES, pH 7.2, 50 NaCl, 1 EGTA) containing 4 µg of
phosphatidylinositol (Sigma), 10 µM ATP, 5 mM MnCl2, and
10 µCi of [-32P]ATP and incubated for 20 min at
30°C. The resulting [32P]phosphatidylinositol
3-phosphate (PIP) lipids were separated from other reaction products by
thin-layer chromatography and were visualized by autoradiography. The
PIP signals were quantitated by densitometry by using the Bio-profile
Bio-1D application (Vilber-Lourmat, France).
Statistical analyses. Data were expressed as means ± SE and analyzed by ANOVA. Differences between treatment groups were analyzed by Duncan's multiple range test.
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RESULTS |
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CLA inhibits proliferation and induces apoptosis of HT-29
cells.
We examined the effect of CLA on viability of HT-29 cells. Cells in
monolayer culture were treated with CLA (0-20 µM) for 2 or 4 days in serum-free medium, and the viable cell number was estimated. As
illustrated in Fig. 1A, CLA
decreased the viable HT-29 cell numbers in a dose-dependent manner with
a 55 ± 2% decrease in cell number 4 days after the addition of
20 µM CLA. To examine whether CLA inhibits DNA synthesis of HT-29
cells, cells in monolayer culture were treated with CLA for 3 days, and
[3H]thymidine incorporation was estimated. As illustrated
in Fig. 1B, CLA decreased the incorporation of
[3H]thymidine into DNA of HT-29 cells in a dose-dependent
manner with a 86 ± 3% decrease in [3H]thymidine
incorporation after the addition of 20 µM CLA.
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CLA regulates ErbB receptor signaling.
ErbB receptors play important roles in regulating epithelial cell
proliferation and survival, so we investigated whether CLA influences
ErbB receptor expression in HT-29 cells. Total cell lysates were
immunoblotted with antibodies specific for EGFR, ErbB2, -3, or -4, and
HT-29 cells were found to express EGFR, ErbB2, and -3 (Fig.
3). ErbB4 was not detectable under the
conditions of the present experiment. Treatment of HT-29 cells with
increasing concentrations of CLA led to decreased EGFR, ErbB2, and -3 levels. HRG was also detected, but CLA did not affect HRG levels.
Immunoblots were probed with an antibody for -actin as a control for
protein loading. To determine whether CLA regulates the expression of EGFR, ErbB2, and -3 at a transcriptional level, cells were similarly treated with CLA, total RNA was isolated, and mRNA levels were determined by RT-PCR analysis. As shown in Fig.
4, CLA decreased EGFR, ErbB2, and ErbB3
transcripts in a concentration-dependent manner.
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HRG--mediated activation of PI3-kinase and Akt is inhibited by
CLA.
Activation of ErbB receptor signaling leads to activation of
PI3-kinase. We examined expression of the p85 regulatory subunit of
PI3-kinase in HT-29 cells treated with increasing amounts of CLA by
using immunoblotting (Fig. 3). Although decreased levels of ErbB
receptors were detected, expression of PI3-kinase was not altered by
the addition of CLA. We investigated whether CLA influences HRG-induced
association of ErbB3 and PI3-kinase. Immunoprecipitations were
performed with an anti-ErbB3 antibody followed by immunoblotting with
the p85 antibody. HRG-
stimulated association of p85 regulatory subunit of PI3-kinase with ErbB3, and the association was reduced in
CLA-treated cells (Fig. 7A).
The levels of PI3-kinase associated with ErbB3 were normalized to ErbB3
expression levels to differentiate whether the reduced association
after CLA treatment is a result of decreased ErbB3 levels or the result
of an inhibition of p85 recruitment. After normalization, the reduced
association after CLA addition is still apparent (Fig. 7B),
indicating that the decrease in PI3-kinase association to ErbB3 is a
result of both decreased ErbB3 levels and an inhibition of p85
recruitment.
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DISCUSSION |
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The anticarcinogenic effect of CLA has been the focus of many recent investigations. We (37) have previously shown that dietary CLA inhibits the development of colon tumors in rats treated with 1,2-dimethylhydrazine. In vitro studies also have shown that CLA inhibits the growth of SW480 (34) and HT-29 cells (35, 42), the human colon cancer lines. The present study indicates that CLA inhibits HT-29 cell growth by both decreasing cell proliferation and inducing apoptosis. However, the magnitude of changes in DNA synthesis due to CLA was much higher than that of apoptotic cell numbers indicating that the growth inhibitory effect of CLA was mainly due to decreased proliferation. Inhibition of proliferation and induction of apoptosis by CLA have already been shown in other cell types. For example, Ip et al. (19) have shown that CLA inhibited proliferation and induced apoptosis of normal mammary epithelial cells, and Palombo et al. (35) reported that trans-10,cis-12 CLA isomer induced caspase-dependent apoptosis in MIP-101 human colorectal carcinoma cells.
In the present study, we provide the first evidence that CLA reduces ErbB2 and ErbB3 protein expression and ErbB3-mediated signaling. When HT-29 cells were incubated with HRG in serum-free medium, the cells responded to HRG by increasing cell number. However, HRG failed to increase the viable cell number when HT-29 cells were treated with 10 µM CLA for 96 h, suggesting that CLA attenuates the HRG-receptor signaling pathway. Indeed, decreased levels of the HRG receptor ErbB3 were noted at 72 h after treatment with CLA. We found that HRG is expressed in HT-29 cells and that the expression of HRG in HT-29 cells was not altered by CLA. Previous studies have shown that HRG is expressed in gastrointestinal tissues (22, 33) and human colon cancer specimens and is an autocrine growth stimulator of GEO human colon cancer cells (48). In addition, production of HRG by mesenchymal cells in the intestine (33) may have an impact on epithelial cells in vivo. HRG may be an autocrine growth regulator of HT-29 cells. However, it is more likely that CLA inhibits cell's ability to respond to HRG without changing the production of this growth factor in HT-29 cells.
CLA decreased ErbB mRNA levels suggesting that CLA regulates the
expression of these receptors at the transcriptional level. The present
study did not determine the mechanisms responsible for the CLA
regulation of ErbB mRNA levels. CLA has been reported to be an
activator of the peroxisome proliferator-activated receptor (PPAR)
(50). CLA or its metabolites may influence transcription of genes that regulate growth by acting as a ligand for the PPAR. Ligands for PPAR
induce apoptosis and exert
antiproliferative effects on several carcinoma cell lines (8,
26).
Activation of tyrosine kinase receptors requires tyrosine
phosphorylation of receptor subunits. HRG induces tyrosine
phosphorylation of ErbB2 and -3 only minimally in cells treated with
CLA, suggesting that CLA inhibits activation of these receptors in
addition to decreasing these receptor protein levels. Observations that
tyrosine phosphorylation of these proteins decreased without changes in protein levels after a short-term treatment with CLA also suggest that
CLA has a direct influence on ErbB2/ErbB3 signaling. Recent studies
utilizing MCF-7 breast cancer cells have shown that the activation of
PPAR through the 15-deoxy-
-12,14-prostaglandin J2 ligand causes a
dramatic inhibition of ErbB2 and -3 tyrosine phosphorylation induced by
HRG-
and HRG-
(38). It remains to be determined
whether CLA inhibits expression of these receptor transcripts and/or
tyrosine phosphorylation of these receptor proteins by activating
PPAR
.
CLA decreased the protein levels of the ErbB receptors in HT-29 cells in a dose-dependent manner. HRG is known to induce the formation of heterodimers between ErbB3 and -2 or between ErbB4 and -2, thereby transactivating ErbB2 (5, 39, 43). The ErbB2/ErbB3 dimer constitutes a high-affinity coreceptor for HRG (43, 44), which is capable of powerful mitogenic signaling (5). Because we observed that ErbB4 was undetectable in HT-29 cells and HRG induced phosphorylation of ErbB2 and -3 in HT-29 cells, it is reasonable to assume that the two receptors form heterodimers.
ErbB3 has been characterized as a major mediator of HRG-dependent activation of the PI3-kinase pathway (5, 44). ErbB3 is particularly well adapted to mediate PI3-kinase signaling because it contains six YXXM consensus-binding sites for p85 (16). We observed that HRG stimulated the recruitment of PI3-kinase to the ErbB3 receptor in HT-29 cells, and CLA decreased ErbB3-associated PI3-kinase protein levels and PI3-kinase activities. These decreases do not appear to be attributed to changes in the PI3-kinase protein expression but to the decrease in ErbB3 protein levels and p85 recruitment.
Akt is a downstream target of PI3-kinase and plays a central role in
PI3-kinase-mediated protection against apoptosis
(9). The present data show that CLA inhibited
HRG--induced activation of Akt, which could be due to both decreased
ErbB2 and -3 levels and decreased ErbB2/ErbB3 activation. The slight
decrease in Akt protein levels may also have contributed to the
decreased p-Akt levels. These results imply that CLA inhibits DNA
synthesis and induces apoptosis of HT-29 cells by inhibiting
the Akt signaling pathway. Increased expression and/or activity of ErbB
receptors and downstream signaling proteins, such as Akt, are frequent
events in cancer. Future studies are needed to examine the effect of Akt on HT-29 cell growth.
In conclusion, we have demonstrated that CLA negatively regulates levels of ErbB receptors and subsequent activation of Akt but not the MAPK pathway in HT-29 cells. Inhibition of ErbB receptor signaling may be one of the mechanisms by which CLA inhibits cancer cell growth and viability. Aberrant activation of ErbB receptors and Akt signaling contributes to the development of many types of cancer, and downregulation of these pathways by CLA could have important therapeutic benefits.
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ACKNOWLEDGEMENTS |
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This study was supported by Korea Health 21 R&D Project, Ministry of Heath and Welfare, Republic of Korea Grant 02-PJ1-PG10-22003-0001.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. H. Y. Park, Division of Life Sciences, Hallym University, 1 Okchon Dong, Chunchon, Korea 200-702 (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.
First published February 5, 2003;10.1152/ajpgi.00347.2002
Received 19 August 2002; accepted in final form 29 January 2003.
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