Activation of PPAR{gamma} by curcumin inhibits Moser cell growth and mediates suppression of gene expression of cyclin D1 and EGFR

Anping Chen1,2 and Jianye Xu1

Departments of 1Pathology and 2Cellular Biology and Anatomy, Louisiana State University Health Sciences Center in Shreveport, Shreveport, Louisiana

Submitted 6 May 2004 ; accepted in final form 9 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Colorectal cancer is a leading cause of cancer-related morbidity and mortality in the United States. Curcumin, the yellow pigment in turmeric, possesses inhibitory effects on growth of a variety of tumor cells by reducing cell proliferation and inducing apoptosis. Effects of the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) on stimulating cell differentiation and on inducing cell cycle arrest have attracted attention from the perspective of treatment and prevention of cancer. The aim of this study was to elucidate the mechanisms by which curcumin inhibits colon cancer cell growth. In the present report, we observed that curcumin, in a dose-dependent manner, inhibited the growth of Moser cells, a human colon cancer-derived cell line, and stimulated the trans-activating activity of PPAR{gamma}. Further studies demonstrated that activation of PPAR{gamma} was required for curcumin to inhibit Moser cell growth. Activation of PPAR{gamma} mediated curcumin suppression of the expression of cyclin D1, a critical protein in the cell cycle, in Moser cells. In addition, curcumin blocked EGF signaling by inhibiting EGF receptor (EGFR) tyrosine phosphorylation and suppressing the gene expression of EGFR mediated by activation of PPAR{gamma}. In addition to curcumin reduction of the level of phosphorylated PPAR{gamma}, inhibition of cyclin D1 expression played a major and significant role in curcumin stimulation of PPAR{gamma} activity in Moser cells. Taken together, our results demonstrated for the first time that curcumin activation of PPAR{gamma} inhibited Moser cell growth and mediated the suppression of the gene expression of cyclin D1 and EGFR. These results provided a novel insight into the roles and mechanisms of curcumin in inhibition of colon cancer cell growth and potential therapeutic strategies for treatment of colon cancer.

colon cancer; receptors; phytochemicals; gene expression; chemoprevention


COLORECTAL CANCER IS A LEADING cause of cancer-related morbidity and mortality in the United States. Epidemiologic data have suggested that dietary modification might reduce the risk of colon cancer by as much as 90% (10, 48). A large amount of studies have focused on development of nontoxic natural agents with preventive and therapeutic activities against colorectal cancer (58). Curcumin, the active ingredient of the rhizome of the plant turmeric (Curcuma longa Linn), is one of the most extensively investigated phytochemicals. Curcumin possesses antiproliferative, antioxidant, anti-inflammatory, and antiangiogenic effects (15, 27, 42). Curcumin has been widely used as a spicy dietary supplement and as an anti-inflammatory medicine in India and China for centuries without apparent adverse effects (58). Epidemiologic studies (43) have indicated that the incidence of large and small bowel adenomas and cancer is relatively low in Indians. The high level of curcumin consumption in India perhaps contributes to the low rate of colon cancer (43). Extensive research over the last 50 years suggests that curcumin could prevent and treat cancer (1). The anticancer potential of curcumin might result from its ability to suppress cell proliferation and induce apoptosis of a wide variety of tumor cells. However, underlying mechanisms of the action are largely as yet to be defined.

Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors (23). It consists of three genes that give rise to three different subtypes (i.e., PPAR{alpha}, -{delta}, and -{gamma}) (20, 21). Among them, PPAR{gamma} is the most widely studied (4). After binding to a ligand, PPAR{gamma} forms heterodimers with the retinoid X receptor and binds to a peroxisome proliferator response element (PPRE) in a gene promoter, leading to regulation of the gene transcription (22). Phosphorylation of PPAR{gamma} by MAPK (ERK and/or JNK) results in inactivation of the receptor and negatively regulates its transcriptional activity (8, 9). Effects of PPAR{gamma} on controlling the expression of genes involved in differentiation and the cell cycle have attracted attention from the perspective of prevention of cancer (49). PPAR{gamma} is highly expressed in adipose tissue, as well as a number of epithelial tissues, including the colonic mucosa (39). The presence of PPAR{gamma} has also been demonstrated in many colon cancer cell lines (29). Activation of PPAR{gamma} by its agonists induces growth arrest and differentiation markers of human colon cancer cells, including Moser and HT-29 (24, 29). Loss-of-function mutations of PPAR{gamma} have been detected in some patients with adenocarcinoma of the colon (54), suggesting that PPAR{gamma} could function as a tumor suppressor. Studies (5) suggested that activation of PPAR{gamma} by its agonists could overcome the anti-apoptotic role of growth hormones in colon cancer cells. However, the role of PPAR{gamma} activation in the development of colon cancer is still controversial (6, 29, 32, 34, 51, 53, 59).

Colonic epithelial homeostasis is maintained by regulated proliferation, differentiation, and apoptosis. Once out of control, these processes could lead to carcinogenesis. These processes are largely controlled by extracellular stimuli, including growth factors. For instance, on binding by its ligand EGF or transforming growth factor-{alpha} (TGF-{alpha}), the EGF receptor (EGFR/erbB1/HER1) is autophosphorylated on tyrosine residues. The EGF signal is propagated by signaling cascades and activation of specific kinases, including c-Raf1, MEK1, and ERK1/2, which could result in diverse effects including cell migration, maturation, differentiation, metastasis, angiogenesis, and inhibition of apoptosis (63). On the other hand, the cell progression through the cell cycle is pivotally controlled by the cyclins through regulation of the activity of cyclin-dependent kinases (47). Cyclin D1 plays a key role in G1/S phase cell cycle progression during cell proliferation (38). The overexpression of EGFR and/or cyclin D1 has been found in a variety of human tumors, including colorectal cancer (18, 28, 52). Previous studies showed that curcumin blocked EGF signaling (19, 30, 31) and inhibited the expression of cyclin D1 in cancer cells (44, 45, 56). The underlying mechanisms remain, however, largely to be defined.

The aim of this study is to elucidate mechanisms of curcumin inhibition of colon cancer cell growth. We have previously demonstrated that curcumin activates PPAR{gamma} in passaged hepatic stellate cells (62). In addition, activation of PPAR{gamma} induces growth arrest and differentiation markers of human colon cancer cells (24, 29). These prior results prompted us to hypothesize that inhibition of colon cancer cell growth by curcumin might be mediated by activation of PPAR{gamma}. In the present report, we demonstrated, for the first time, that curcumin activated PPAR{gamma} in Moser cells, a human colon cancer-derived cell line, leading to inhibition of cell growth by inhibiting tyrosine phosphorylation of EGFR and suppressing the gene expression of EGFR and cyclin D1. Activation of PPAR{gamma} was required for curcumin to suppress the expression of EGFR and cyclin D1 genes. The phytochemical stimulated PPAR{gamma} trans-activating activity mainly by inhibiting cyclin D1 expression. These results provided a novel insight into the roles and mechanisms of curcumin in inhibition of colon cancer cell growth.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Material and cell culture. Moser cells, a human colon carcinoma cell line, was a gift from Dr. Michael G. Brattain (Dept. of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY) (35). Cells were cultured in modified McCoy's 5A medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. HT-29 cells, purchased from American Type Culture Collection (Rockville, MD), were cultured in modified McCoy's 5A medium supplemented with 10% FBS. Curcumin (purity: >94%) was purchased from Sigma (St. Louis, MO). PD-68235, a specific PPAR{gamma} antagonist (7), was kindly provided by Pfizer (Ann Arbor, MI). PD-98059 and SP-650012 were purchased from Pfizer. PGJ2 was purchased from Biomol Research Lab (Plymouth Meeting, PA).

Determination of cell growth. Preconfluent Moser cells (2–3 days postplating) were treated with PD-68235 at 10 µM for 30 min before the addition of curcumin or PGJ2 at indicated concentrations for an additional 24, 48, or 72 h. Cell growth was determined by either MTS assays or cell numbers. For MTS assays, the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay kit was used after the protocol provided by the manufacturer (Promega) (14). MTS assay is a colorimetric method for determining the number of viable cells. For counting cell numbers, after being washed with cold PBS, cells were briefly treated with trypsin. Cell numbers were counted by a computer-equipped cell counter (Coulter, Miami, FL). Each treatment was done in triplicate and repeated for at least three times (62).

Lactate dehydrogenase release assays. Lactate dehydrogenase (LDH) assays were performed as recently described (62). In brief, preconfluent Moser cells were treated with curcumin at the indicated concentrations for 24 or 72 h without changing media. LDH in conditioned medium was determined as medium LDH. LDH in cell lysates was analyzed as cellular LDH. LDH in DMEM with 10% FBS was defined as contamination arising from FBS and subtracted from medium and cellular LDH. LDH activities were determined with the use of a Cytoscan-LDH Cytotoxicity Assay kit (Geno Technology, St. Louis, MO). Results were shown as percentage of total LDH, i.e., medium LDH%/(medium LDH + cellular LDH). Values were expressed as means ± SD (n ≥ 3).

Plasmids and transient transfection assays. The PPAR{gamma} reporter plasmid pPPRE-TK-Luc contains three copies of peroxisome proliferator response element (PPRE) from acyl-CoA oxidase gene linked to the herpes virus thymine kinase (TK) promoter (–105/+51) subcloned in a luciferase vector, a gift from Dr. Kevin J. McCarthy (Louisiana State University Health Sciences Center, Shreveport, LA) (40, 50). The EGFR luciferase reporter plasmid pER1 was a gift from Dr. Alfred C. Johnson (Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD). The plasmid pCMV-cyclin D1 containing cyclin D1 cDNA was a gift from Dr. Richard G. Pestell (Department of Oncology, Lombardi Cancer Center, Georgetown Univ., Washington, DC). Semiconfluent cells in six-well plastic plates were transiently transfected by using the Lipofectamine reagent (Life Technologies, Grand Island, NY). Each sample (total 3–4 µg DNA/well) was done in triplicate in every experiment. Luciferase assays were performed as previously described (11, 12, 14, 62). Transfection efficiency was determined by cotransfection of a {beta}-galactosidase reporter, pSV-{beta} gal (0.5 µg/well) (Promega). {beta}-Galactosidase activity was measured by a chemiluminescence assay kit (Tropix, Bedford MA) according to the manufacturer's instructions. Each experiment was independently repeated for at least three times. Luciferase activities were normalized by {beta}-galactosidase activities in cells and were averaged among experiments. Transfection results were expressed in relative luciferase activity as means ± SD.

Immunoprecipitation assays. Cells treated with or without curcumin at indicated concentrations for 24 h were lysed with RIPA buffer. To detect phosphorylated PPAR{gamma}, 50 µg of whole cell protein extracts were incubated with anti-PPAR{gamma} antibodies (1 µg) (Santa Cruz Biotechnology, Santa Cruz, CA) in 500 µl of RIPA buffer at 4°C overnight. After incubating with 50 µl of protein A-Sepharose at 4°C for 4 h, each sample was washed with RIPA buffer for three times and then twice in cold PBS. Samples were resuspended and boiled in 30 µl of loading buffer. Phosphorylated PPAR{gamma} was separated and detected by Western blot analyses using anti-phosphoserine antibodies (Chemicon, Temecula, CA). The level of phospho-PPAR{gamma} in the total PPAR{gamma} was determined by using Quantity One 4.4.1 (Bio-Rad, Hercules, CA).

Western blot analyses. Whole cell protein extracts were prepared from preconfluent Moser cells treated with or without curcumin. SDS/PAGE with 10% resolving gel was used to separate proteins (25 µg/lane). The separated proteins were detected by using primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Protein bands were visualized by using chemiluminescence reagent (Kirkegaard and Perry Laboratories, Gaithersburg, MD). After normalization with the internal control {beta}-actin, the level of phospho-PPAR{gamma} in the total PPAR{gamma} was determined by using Quantity One 4.4.1 (Bio-Rad).

RNA isolation and real-time PCR. Total RNA was isolated by TRI-Reagent (Sigma), following the protocol provided by the manufacturer. Real-time PCR was carried out as previously described (14, 62). In brief, DNase I-treated total RNA (1 µg) was used for synthesis of the first strand of cDNA. Reverse transcription conditions were as follows: 42°C for 15 min, 95°C for 5 min, and 5°C for 5 min (one cycle). Real-time PCR was carried out in 25 µl of reaction solution [2.5 µl of 10 x buffer, 5 mM 2-deoxynucleotide 5'-triphosphate, 10 mM MgCl2, 200 nM primers, and 0.75 units of platinum Taq polymerase (all from Invitrogen) plus 1 µl of SYBR Green (1:2,000; BioWhittaker, Richland, ME)]. No genomic DNA contamination or pseudogenes were detected by PCR without the reverse-transcription step in the total RNA used. Reactions started at 95°C for 7 min, followed by 40 cycles of 95°C for 20 s, 54°C for 30 s, and 72°C for 30 s. Melting peaks of PCR products were determined by heat-denaturing them over a 35°C temperature gradient at 0.2°C/s from 60 to 95°C. mRNA fold changes in target genes relative to the {beta}-actin control were calculated as suggested by Schmittgen et al. (55). Primers used in real-time PCR were: EGFR: (forward) 5'-GTG ACC GTT TGG GAG TTG ATG A-3', (reverse) 5'-GGC TGA GGG AGG CGT TCT C-3'; cyclin D1: (forward) 5'-CTG GAT GCT AGA GGT CTG CAGE-3'; (reverse) 5'-AGA GAS AGAR ARC COG TCC AGG-3'; {beta}-actin: (forward) 5'-GGG GGA AAT GGT GGG TGA CAT-3', (reverse) 5'-GAT GGA GTT GAA GGT AGT TTC-3'.

Statistical analysis. Differences between means were evaluated by using an unpaired two-sided Student's t-test (P < 0.05 was considered significant). Where appropriate, comparisons of multiple treatment conditions with control were analyzed by ANOVA with Dunnett's test for post hoc analysis.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of PPAR{gamma} is required for curcumin to inhibit Moser cell growth. Moser cells, a human colon cancer-derived cell line, were previously demonstrated to be suitable for studying gene regulation and gene expression in responding to activation of PPAR{gamma} by two different synthetic agonists (24). These prior studies enable us to use the cell line to avoid loss-of-function mutations in PPAR{gamma} (54). To evaluate the effect of curcumin on cell growth, Moser cells were treated with curcumin (15 µM) for 24, 48, or 72 h, respectively. The effect of curcumin on cell growth was determined by MTS assays (Fig. 1A), a colorimetric method for determining the number of viable cells, as well as by counting total cell numbers (data not shown). As shown in Fig. 1A, compared with the no-treatment control, treatment of cells with curcumin for 24 h caused a significant reduction in viable cell numbers by 13%, indicating that curcumin caused a reduction in cell growth. Similarly, PGJ2 (5 µM), a natural PPAR{gamma} agonist, reduced the viable Moser cell number by 12% (Fig. 1A), suggesting that activation of PPAR{gamma} could result in inhibition of Moser cell growth. To evaluate the role of PPAR{gamma} in the inhibitory effect of curcumin, Moser cells were pretreated with the specific PPAR{gamma} antagonist PD-68235 (10 µM) for 30 min before the addition of curcumin or PGJ2 for an additional 1, 2, or 3 days. MTS assays demonstrated that pretreatment of cells with the PPAR{gamma} antagonist apparently abrogated the inhibitory effect of curcumin or PGJ2 on cell growth (Fig. 1A). This result suggested that curcumin might activate PPAR{gamma}, which was required for inhibition of Moser cell growth by curcumin or PGJ2. PD-68235 itself stimulated cell growth by 13% after 24 h, presumably by blocking endogenous PPAR{gamma} activity, which supported the theory of the inhibitory role of PPAR{gamma} activation in Moser cell growth.



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Fig. 1. Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) is required for curcumin to inhibit Morse cell growth, MTS cell growth assays of preconfluent Moser cells treated with curcumin with or without the specific PPAR{gamma} antagonist PD-68235 (PD; 10 µM). Values were expressed as means ± SD (n ≥ 6). A: Moser cells were pretreated with or without PD-68235 (10 µM) for 30 min before the addition of curcumin (Cur) at 15 µM, or PGJ2 (5 µM), a natural PPAR{gamma} ligand, for an additional 24, 48, or 72 h. Numbers were alterations in cell growth after 24-h treatment compared with cells with no treatment for 24 h. *P < 0.05, vs. cells with no treatment. {dagger}P < 0.05 vs. cells treated with only curcumin or PGJ2 for a corresponding time. B: Moser cells were treated with curcumin at indicated concentrations for 24 h with or without PD-68235 pretreatment (10 µM). *P < 0.05 vs. cells with no treatment. {dagger}P < 0.05 vs. cells treated with only curcumin at the same concentration.

 
To verify the necessity of PPAR{gamma} activation in curcumin inhibition of Moser cell growth, additional cell growth assays were carried out. Moser cells were treated with curcumin at indicated concentrations for 24 h with or without PD-68235 pretreatment (10 µM). As shown in Fig. 1B, curcumin caused a dose-dependent reduction in cell growth. The inhibitory effect of curcumin at each concentration tested was, at least partially, eliminated by pretreatment of Moser cells with PD-68235 (Fig. 1B), suggesting that activation of PPAR{gamma} might play a critical role in curcumin inhibition of Moser cell growth. Similar results were obtained when cell growth was determined by counting total cell numbers (data not shown). Curcumin toxicity to Moser cells was carefully studied by examining LDH release. As shown in Table 1, curcumin compared with the control made no significant difference in LDH release at concentrations within 20 µM. The curcumin dose at 30 µM and higher might cause toxicity to Moser cells. On the basis of this observation and a rapid recovery of cell proliferation after withdrawal of curcumin (data not shown) and trypan blue exclusion assays (data not shown), it was concluded that curcumin at 20 µM or less was not toxic to Moser cells. The reduction in viable cell numbers by curcumin at concentrations higher than 30 µM in Fig. 1B might be caused not only by inhibition of cell growth, but also by toxicity, which was unlikely rescued by the PPAR{gamma} antagonist. Therefore, the concentration of curcumin used in the following studies was no greater than 20 µM.


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Table 1. LDH release in Moser cell treated with curcumin

 
Curcumin stimulates the trans-activating activity of PPAR{gamma} in Moser cells. Further experiments were conducted to verify the effect of curcumin on stimulating the trans-activating activity of PPAR{gamma}. Moser cells were transfected with the PPAR{gamma} reporter plasmid pPPRE-TK-Luc, containing three copies of PPAR-response elements inserted into a luciferase reporter vector. After recovery, cells were pretreated with or without PD-68235 for 30 min before the addition of curcumin or PGJ2 at the indicated concentrations for an additional 36 h. Luciferase assays demonstrated that compared with control, curcumin like PGJ2 caused significant increases in luciferase activity in a dose-dependent manner (Fig. 2). Cells treated with both curcumin and PGJ2 showed a further significant increase in luciferase activity. Pretreatment of cells with the PPAR{gamma} antagonist PD-68235 significantly reduced luciferase activity induced by curcumin. Without curcumin, PD-68235 itself reduced luciferase activity, suggesting that the PPAR{gamma} antagonist might block the activity of endogenous PPAR{gamma} in these cells. Taken together, these results demonstrated that curcumin stimulated the trans-activating activity of PPAR{gamma} in Moser cells. PPAR{gamma} ligands are presumed to exist in the medium with 10% FBS (25, 41).



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Fig. 2. Curcumin stimulates the trans-activating activity of PPAR{gamma} in Moser cells. Preconfluent Moser cells were transiently transfected with the PPAR{gamma} reporter plasmid pPPRE-TK-Luc containing 3 copies of peroxisome proliferator response element (PPRE) in a luciferase reporter vector. After recovery, cells were pretreated with or without PD-68235 for 30 min before the addition of curcumin or PGJ2 at indicated concentrations for an additional 36 h. Luciferase activities were expressed as relative units after {beta}-galactosidase normalization. Values were expressed as means ± SD (n = 6). *P < 0.05 vs. cells with no treatment. **P < 0.05 vs. cells treated with the same dose of curcumin only.

 
Curcumin treatment causes the reduction in the level of phosphorylated PPAR{gamma} in Moser cells. To elucidate the underlying mechanisms of activation of PPAR{gamma} by curcumin, we initially hypothesized that curcumin might regulate the expression of PPAR{gamma}. To test the hypothesis, Moser cells were treated with or without curcumin at 15 µM for 24 h. Whole cell protein extracts were prepared from these cells. As shown in Fig. 3A, results from Western blot analyses, however, did not support our initial hypothesis. Curcumin did not change the abundance of PPAR{gamma} in Moser cells (Fig. 3A). In fact, a large amount of PPAR{gamma} was easily detected in Moser cells. Although the abundance of PPAR{gamma} was not changed, an upper protein band was significantly reduced by approximately four- to fivefold by curcumin as exemplified in Fig. 3A. Previous studies indicated that PPAR{gamma} could exist in two forms regarding the phosphorylation status (8, 9). Phosphorylation of PPAR{gamma} by ERK and/or JNK results in inactivation of the receptor, which shows no transcriptional activity even after binding by its agonist (8, 9). To examine the effect of curcumin on the phosphorylation status of the receptor, immunoprecipitation assays were performed. Total PPAR{gamma} was immunoprecipitated from the above protein extracts using anti-PPAR{gamma} antibodies. Phosphorylated PPAR{gamma} was detected by Western blot analyses using anti-phosphoserine antibodies. As shown in Fig. 3B, curcumin significantly reduced the abundance of phosphorylated PPAR{gamma} by approximately four- to fivefold in Moser cells, suggesting that curcumin treatment might cause a reduction in the level of phosphorylated PPAR{gamma} in Moser cells.



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Fig. 3. Curcumin results in reduction in the level of phosphorylated PPAR{gamma} (p-PPAR{gamma}) in Moser cells. A: Western blot analyses of Moser cells treated with or without curcumin at 15 µM for 24 h using anti-PPAR{gamma} antibodies. Representatives of 3 independent experiments are shown here. After normalization with the control {beta}-actin, the level of p-PPAR{gamma} in the total PPAR{gamma} was determined and expressed as means ± SD (n = 3). *P < 0.05 vs. cells with no curcumin treatment. B: to study the effect of curcumin on the phosphorylation status of PPAR{gamma}, immunoprecipitation assays of the above cell extracts were performed by using anti-PPAR{gamma} antibodies and antiphosphoserine antibodies in order. Total PPAR{gamma} was used as a control for equal protein loading. After normalization with the total PPAR{gamma}, the effect of curcumin on reducing the level of p-PPAR{gamma} was determined and expressed as means ± SD (n = 3). *P < 0.05 vs. cells with no curcumin treatment.

 
Blocking the ERK and/or JNK signal pathways results in an increase in PPAR{gamma} activity. We and others (13, 16, 26) have previously reported that curcumin effectively inhibits ERK and JNK activities in a variety of cells, including colon cancer cells. To further demonstrate the effect of ERK and JNK on curcumin inhibition of PPAR{gamma} activity, Moser cells were transiently transfected with the PPAR{gamma} reporter plasmid pPPRE-TK-Luc. Cells were then treated for 36 h with curcumin, or PD-98059, a specific ERK inhibitor, and/or SP-650012, a specific JNK inhibitor. As shown in Fig. 4, luciferase assays demonstrated that curcumin at 15 µM resulted, as expected, in a significant increase in luciferase activity in these cells. Compared with no treatment, PD-98059 or SP-650012, like curcumin, significantly increased luciferase activity in the cells, as well (Fig. 4). Combination of PD-98059 and SP-650012 showed, however, no synergic effects on luciferase activity. These results indicated that blocking the ERK or JNK signal pathways increased the trans-activating activity of endogenous PPAR{gamma} in Moser cells. Taken together, these results suggested that inhibition of ERK and/or JNK signal pathways by curcumin might contribute to the activation of PPAR{gamma} presumably by inhibiting the extent of PPAR{gamma} phosphorylation. However, it is noteworthy that because the amount of phosphorylated PPAR{gamma} stood for a small portion in the total PPAR{gamma} (~5%, Fig. 3), the reduction in the level of phosphorylated PPAR{gamma} unlikely played a major role in curcumin stimulation of the trans-activating activity of the receptor. Further experiments were performed to elucidate the additional mechanisms.



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Fig. 4. Inhibition of MAPK activity by curcumin leads to activation of PPAR{gamma}. To demonstrate the effect of ERK and JNK on inhibition of PPAR{gamma} trans-activity, Moser cells were transiently transfected with pPPRE-TK-Luc. Cells were then treated for 36 h with either curcumin (15 µM) or PD-98059 (25 µM), a specific ERK inhibitor, and/or SP-650012 (10 µM), a specific JNK inhibitor. Luciferase activities were expressed as relative units after {beta}-galactosidase normalization. Values were expressed as means ± SD (n = 6). *P < 0.05 vs. cells with no treatment.

 
Activation of PPAR{gamma} mediates the suppression of the cyclin D1 expression by curcumin, which in turn facilitates the activation of PPAR{gamma}. Previous studies demonstrated that curcumin inhibited cyclin D1 gene expression, resulting in cell cycle arrest (44, 45, 56). The underlying mechanisms remain largely unknown. We hypothesized that activation of PPAR{gamma} might mediate the suppression of cyclin D1 expression by curcumin. To test the hypothesis, preconfluent Moser cells were pretreated with or without PD-68235 (10 µM) for 30 min before the addition of curcumin at indicated concentrations for an additional 24 h. Total RNA or protein extracts were prepared for real-time PCR (Fig. 5A) or Western blot analyses (Fig. 5B), respectively. Experimental results indicated that cyclin D1 was highly expressed in Moser cells, which was significantly inhibited at both transcriptional and translational levels by curcumin in a dose-dependent manner (Fig. 5, A and B). Pretreatment of cells with the PPAR{gamma} antagonist PD-68235 abrogated the inhibitory effect, suggesting that activation of PPAR{gamma} might be required for curcumin to inhibit the gene expression of cyclin D1 in Moser cells.



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Fig. 5. Activation of PPAR{gamma} is required for curcumin suppression of cyclin D1 expression, which in turn relieves inhibition of PPAR{gamma} activity and facilitates curcumin activation of PPAR{gamma}. Preconfluent Moser cells were pretreated with or without PD-68235 (10 µM) for 30 min before the addition of curcumin at indicated concentrations for 24 h. A: total RNA was prepared for real-time PCR. mRNA fold changes were calculated by using {beta}-actin as a control (see MATERIALS AND METHODS for details). Values were expressed as means ± SD from 3 independent experiments. *P < 0.05 vs. cells with no treatment. **P < 0.05 vs. cells treated with curcumin (15 µM) only. B: whole cell protein extracts were prepared for Western blot analyses. {beta}-Actin was used as an internal control for equal protein loading. Representative of 3 independent experiments was presented. C: to evaluate the effect of cyclin D1 on inhibition of PPAR{gamma} activity, Moser cells in 6-well culture plates were cotransfected with a total of 4.5 µg/well of plasmid DNA, including 2 µg of pPPRE-TK-Luc, 0.5 µg of {beta}-galactosidase reporter, pSV-{beta} gal, pCMV-cyclin D1 at indicated doses, and the empty vector pcDNA. The amount of DNA of pCMV-cyclin D1 plus pcDNA was equal to 2 µg. Cells were then treated with or without curcumin at 15 µM for 36 h. Luciferase activities were expressed as relative units after {beta}-galactosidase normalization. Values were expressed as means ± SD (n = 6). {dagger}P < 0.05 vs. cells without curcumin and pCMV-cyclin D1 in transfection (first from left); *P < 0.05 vs. cells with curcumin but without pCMV-cyclin D1 in transfection (5th column from left).

 
Other studies have previously indicated that overexpression of cyclin D1 inhibits the ligand-induced activation of PPAR{gamma} (61). We therefore hypothesized that in addition to the reduction in the level of phosphorylated PPAR{gamma}, inhibition of cyclin D1 expression by curcumin might also contribute to and even play a major role in activation of the receptor because the majority of PPAR{gamma} were not phosphorylated (Fig. 3). If that was the case, we further inferred that increasing the expression of cyclin D1 should abrogate the stimulatory effect of curcumin on PPAR{gamma} activation in Moser cells. To study this hypothesis, Moser cells in six-well culture plates were cotransfected with the PPAR{gamma} reporter plasmid pPPRE-TK-Luc and the cyclin D1 expressing plasmid pCMV-cyclin D1, containing cyclin D1 cDNA at indicated doses. A total of 4.5 µg of plasmid DNA was used in each well for the transfection, including 2 µg of pPPRE-TK-Luc, 0.5 µg of pSV-{beta} gal, and pCMV-cyclin D1 at the indicated doses, and the empty vector pcDNA. The amount of DNA of pCMV-cyclin D1 plus pcDNA was equal to 2 µg. After transfection, cells were treated with or without curcumin at 15 µM for 36 h. As shown in Fig. 5C, luciferase assays demonstrated that without curcumin, the increase in the cyclin D1 cDNA itself led to a dose-dependent reduction in luciferase activity, suggesting that forced expression of cyclin D1 suppressed the activity of endogenous PPAR{gamma} in the basal state in Moser cells. Curcumin treatment dramatically increased, as expected, PPAR{gamma} activity demonstrated by an increase in luciferase activity. Along with the increase in the dose of pCMV-cyclin D1, luciferase activity induced by curcumin was gradually diminished (Fig. 5C), indicating that forced expression of cyclin D1 could block the activation of PPAR{gamma} induced by curcumin in Moser cells. Taken together, these results demonstrated that activation of PPAR{gamma} by curcumin was required for suppression of the cyclin D1 gene expression, which might in turn facilitate the activation of PPAR{gamma} in Moser cells. These results also suggested that high expression of cyclin D1 in Moser cells might inhibit the activity of PPAR{gamma} in the basal state.

Curcumin inhibits tyrosine-phosphorylation of EGFR in Moser cells. Activation of EGF signaling results in diverse effects, including stimulation of cell growth and inhibition of apoptosis (63). In addition to the effect of the reduction in cyclin D1 expression on curcumin inhibition of Moser cell growth, it was further hypothesized that curcumin might also block EGF signaling in Moser cells. To test the hypothesis, studies first focused on evaluating the effect of curcumin on tyrosine phosphorylation of EGFR. To reduce endogenous phosphorylated tyrosines in EGFR, semiconfluent Moser cells were serum starved in serum-free medium for 24 h. Cells were then pretreated with or without curcumin at 15 µM for 30 min before the addition of EGF (50 ng/ml). Cells were harvested at the indicated time points and prepared for Western blot analyses using antibodies against phospho-EGFR (Santa Cruz Biotechnology). As shown in Fig. 6, EGF rapidly activated EGFR by phosphorylation in <1 min (lane 2), with maximal activation at ~10 min (lane 8). The level of phosphorylated EGFR was declining by 30 min (Fig. 6, lane 10). Curcumin significantly inhibited the phosphorylation of EGFR at the time points tested (Fig. 6, lanes 3, 5, 7, 9, and 11). Other than EGF, some components in serum, including lysophosphatidic acid, could trans-activate EGFR after binding to its G protein-coupled receptor (33, 37). Further experiments were therefore performed to evaluate the effect of curcumin on EGFR phosphorylation stimulated by 10% FBS. It was observed that curcumin similarly inhibited EGFR phosphorylation induced by serum in Moser cells (data not shown). Taken together, these experiments demonstrated that curcumin inhibited tyrosine-phosphorylation of EGFR induced by EGF and/or serum in Moser cells.



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Fig. 6. Curcumin inhibits tyrosine-phosphorylation of EGFR induced by EGF in Moser cells. Semiconfluent Moser cells were serum-starved in serum-free medium for 24 h. Cells were then pretreated with or without curcumin at 15 µM for 30 min before addition of EGF (50 ng/ml). Cells were harvested at the indicated times and prepared for Western blot analyses using antibodies against phospho-EGFR. {beta}-Actin was used as an internal control for equal protein loading. Representation of 3 independent experiments was presented.

 
Activation of PPAR{gamma} is required for curcumin to suppress the expression of EGFR in Moser cells. To evaluate the effect of curcumin on regulating EGFR gene expression, Moser cells were treated with curcumin at indicated concentrations for 8 h. Total proteins or RNA extracts were prepared for Western blot analysis (Fig. 7A) or real-time PCR (Fig. 7B), respectively. Experimental results demonstrated that curcumin, in a dose-dependent manner, significantly reduced the abundance of EGFR protein (Fig. 7A) and the steady-state level of EGFR mRNA (Fig. 7B). Pretreatment of cells with PD-68235 (10 µM) for 30 min apparently abrogated the inhibitory effect (Fig. 7, A and B), indicating that activation of PPAR{gamma} was required for curcumin inhibition of EGFR expression in Moser cells. To evaluate the effect of curcumin on the promoter of the gene, Moser cells were transfected with the EGFR luciferase reporter plasmid pER1, in which a fragment of EGFR promoter region (1109 bp nucleotides) was subcloned into a luciferase reporter plasmid. After transfection, cells were pretreated with or without PD-68235 (10 µM) for 30 min before the addition of curcumin at indicated concentrations for an additional 8 h. As shown by luciferase assays in Fig. 7C, curcumin caused a dose-dependent reduction in luciferase activity, suggesting the inhibitory effect of curcumin on the gene promoter of EGFR, which was eliminated by PD-68235 pretreatment (Fig. 7C). Taken together, these results demonstrated that curcumin caused dose-dependent inhibition of EGFR gene expression in Moser cells, which required the activation of PPAR{gamma}.



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Fig. 7. Activation of PPAR{gamma} mediates curcumin suppression of the gene expression of EGFR in Moser cells. To study the effect of curcumin on the expression of EGFR and to elucidate the underlying mechanism, preconfluent Moser cells were pretreated with or without PD-68235 (10 µM) for 30 min before addition of curcumin at indicated concentrations for an additional 8 h. A: whole cell protein extracts were prepared for Western blot analyses. {beta}-Actin was used as an internal control for equal protein loading. Representation of 3 independent experiments was presented. B: total RNA was prepared for real-time PCR. mRNA fold changes were calculated by using {beta}-actin as a control. Values were expressed as means ± SD (n = 3). *P < 0.05 vs. cells with no treatment. **P < 0.05 vs. cells treated with curcumin (20 µM) only. C: Moser cells were transfected with the EGFR luciferase reporter plasmid pER1 containing a fragment of the EGFR gene promoter (see MATERIALS AND METHODS for details). After recovery, cells were pretreated with or without PD-68235 (10 µM) for 30 min before addition of curcumin at indicated concentrations for an additional 8 h. Luciferase activities were expressed as relative units after {beta}-galactosidase normalization. Values were expressed as means ± SD (n = 6). *P < 0.05 vs. cells with no treatment. **P < 0.05 vs. cells treated with curcumin (20 µM) only.

 

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Accumulating evidence has demonstrated the inhibitory effects of curcumin on the development of colon cancer (10, 27, 43, 48). The underlying mechanisms remain largely to be defined. In the present study, we demonstrated that curcumin inhibited cell growth of human colon cancer-derived Moser cells, at least partially, by activating PPAR{gamma}, suppressing the expression of cyclin D1, and blocking EGF signaling by inhibiting tyrosine phosphorylation and the gene expression of EGFR. Activation of PPAR{gamma} was required for curcumin to inhibit the expression of cyclin D1 and EGFR genes in Moser cells. Curcumin stimulated the trans-activating activity of PPAR{gamma} mainly by inhibiting the expression of cyclin D1 and relieving the inhibitory effect on PPAR{gamma} activity in the basal state. To study the generality of the curcumin effect on colon cancer cell growth, other human colon cancer cell lines, including HT-29, were studied. Cell proliferation assays demonstrated that curcumin significantly inhibited cell growth of HT-29 (data not shown). This cell line was more sensitive to curcumin than Moser cells regarding the reduction in cell growth. However, blocking PPAR{gamma} with its antagonist was less effective in HT-29 than in Moser cells, suggesting that other mechanisms, in addition to PPAR{gamma} activation, might also contribute to curcumin inhibition of HT-29 cell growth. The present study focused on the role of PPAR{gamma} activation in curcumin inhibition of colon cancer cell growth. Moser cells were previously demonstrated to be suitable for studying the expression of genes in responding to PPAR{gamma} activation (24). Experiments were therefore restricted in Moser cell responses to curcumin treatment in this study. It bears emphasis, however, that our results do not exclude the possible involvement of other mechanisms in curcumin inhibition of colon cancer cell growth. Curcumin at 15 µM was chosen for most of our in vitro experiments, which is higher than those observed in blood and tissues of humans and animals (2, 46). Dietary supplementation is unlikely to achieve comparable concentrations in vivo that are effective in vitro (2), but many differences between in vivo and in vitro experimental systems make direct comparisons problematic.

Our previous studies (62) demonstrated that curcumin activated PPAR{gamma} in rat hepatic stellate cell by inducing the expression of PPAR{gamma}. In the present study, we observed that curcumin caused no alteration in the abundance of PPAR{gamma} in Moser cells. Results from Western and immunoprecipitation assays demonstrated that PPAR{gamma} existed in two forms with or without phosphorylation. Curcumin treatment of cells dramatically reduced the abundance of phosphorylated PPAR{gamma}, suggesting that curcumin might inhibit the extent of PPAR{gamma} phosphorylation and reduce the level of phosphorylated PPAR{gamma} (Fig. 3, A and B). Phosphorylation of PPAR{gamma} by ERK or by JNK negatively regulates the trans-activating activity of the receptor (8, 9). We and others have previously reported that curcumin effectively inhibits ERK and JNK activities in a variety of cells, including colon cancer cells (12, 13, 16, 26, 36). The present report demonstrated that blocking ERK and/or JNK signal pathways by specific inhibitors resulted in an increase in the trans-activating activity of PPAR{gamma} (Fig. 4). Taken together, our results suggested that curcumin inhibition of the extent of PPAR{gamma} phosphorylation might be conducive to the activation of the receptor in Moser cells. However, because a small portion of the total PPAR{gamma} (~5%) was affected (Fig. 3A), curcumin reduction of the level of phosphorylated PPAR{gamma} unlikely played a major role in curcumin stimulation of the activity of PPAR{gamma} in Moser cells.

PPAR{gamma} was highly expressed in Moser cells and the majority of it was in the form without phosphorylation (Fig. 3). However, the trans-activating activity of the receptor was relatively low (Fig. 2). Additional mechanisms likely existed to inhibit the activity of PPAR{gamma} in the basal state in Moser cells. Additional experiments were performed to explore the mechanisms. Prior experiments demonstrated that high expression of cyclin D1 might inhibit PPAR{gamma} activity (61). Our results indicated that the level of cyclin D1 expression was rather high in Moser cells (Fig. 5, A and B). Curcumin significantly reduced the level of cyclin D1 in a dose-dependent manner. It is therefore hypothesized that the majority of PPAR{gamma} activity in the basal state in Moser cells might be suppressed by high expression of cyclin D1. Suppressing the expression of cyclin D1 might play a major role in curcumin stimulation of PPAR{gamma} transcriptional activity in Moser cells. To test the hypothesis, Moser cells were cotransfected with cyclin D1 cDNA and the PPAR{gamma} reporter plasmid. Experimental results demonstrated that forced expression of cyclin D1 inhibited PPAR{gamma} activity in the basal state and abrogated, in a dose-dependent manner, the stimulatory effect of curcumin on PPAR{gamma} trans-activating activity (Fig. 5C). On the other hand, activation of PPAR{gamma} was also required for curcumin to inhibit the expression of cyclin D1 gene (Fig. 5, A and B). This observation was supported by other prior reports that the PPAR{gamma} agonist PGJ2 inhibited the expression of cyclin D1 (60). Taken together, these results support our hypothesis that in addition to the reduction in the level of phosphorylated PPAR{gamma}, inhibition of cyclin D1 expression might play a major and critical role in curcumin activation of PPAR{gamma} in induction of cell cycle arrest and inhibition of Moser cell growth, which is consistent with previous reports (44, 45, 56).

EGFR is an attractive target for the development of cancer therapeutics (3, 17, 18). Inhibition of EGFR expression impairs tumor growth (3, 17, 18). In addition to inhibiting the expression of cyclin D1, a key player in G1/S phase cell cycle progression during cell proliferation (38), blocking EGF signaling might also make an important contribution to curcumin inhibition of Moser cell growth. Our results showed that curcumin possessed dual actions on interfering with the EGF signal pathway. Tyrosine phosphorylation in EGFR serves as a critical link between extracellular EGF stimulation and intracellular signal propagation. Our results demonstrated that curcumin significantly inhibited tyrosine phosphorylation of EGFR elicited by EGF or serum (Fig. 6). This observation was supported by other studies showing the inhibitory effect of curcumin on EGFR kinase activity in human cancer cells, including human epidermoid carcinoma A431 cells and human breast cancer cell line MDA-MB-468 (30, 31, 57). Underlying mechanisms remain poorly understood. In addition to the inhibitory action on tyrosine phosphorylation of the receptor, curcumin also exerted its action on inhibiting EGFR gene expression in Moser cells. Curcumin apparently caused a dose-dependent reduction in the abundance of EGFR and inhibited the expression of EGFR gene in Moser cells (Fig. 7). Our observations were consistent with previous reports that the inhibitory effects of curcumin on the EGFR signaling were accomplished by downregulating the expression of EGFR and inhibiting the endogenous EGFR tyrosine kinase activity (19, 30, 31). In addition, we further observed that inhibition of EGFR expression by curcumin required the activation of PPAR{gamma}. Pretreatment of cells with the PPAR{gamma} antagonist eliminated the inhibitory effect of curcumin on EGFR gene expression (Fig. 7). Further experiments are ongoing to investigate the mechanisms by which activation of PPAR{gamma} by curcumin downregulates the expression of EGFR in colon cancer cells.

On the basis of our observations and results, a model was proposed to explain, at least partially, the mechanisms by which curcumin inhibits colon cancer cell growth, including Moser cells (Fig. 8). Curcumin inhibits Moser cell growth by stimulating PPAR{gamma} activity and suppressing the expression of cyclin D1 and blocking EGF signaling. PPAR{gamma} in Moser cells exists in two forms on the basis of the phosphorylation status. Curcumin inhibits ERK and JNK activities, which might lead to reduction in the level of phosphorylated PPAR{gamma} and to stimulation of PPAR{gamma} activity. Curcumin inhibition of the extent of PPAR{gamma} phosphorylation likely plays a minor role in activation of the receptor in Moser cells, because a small portion of PPAR{gamma} is affected. In contrast, suppression of cyclin D1 gene expression might make a major contribution to curcumin activation of the majority of PPAR{gamma}, as well as to induction of cell cycle arrest. In addition, curcumin effectively blocks EGF signaling and results in reduction of cancer cell growth by inhibiting EGFR tyrosine phosphorylation and suppressing EGFR gene expression. The latter requires the activation of PPAR{gamma}. Further experiments are ongoing to elucidate the mechanisms by which activation of PPAR{gamma} mediates curcumin inhibition of the expression of cyclin D1 and EGFR genes in Moser cells. Additional experiments are necessary to determine other mechanisms and signal pathways involved in curcumin inhibition of colon cancer cell growth, including induction of cell cycle arrest and apoptosis. In summary, our results demonstrated for the first time that curcumin activation of PPAR{gamma} inhibited Moser cell growth and mediated the suppression of the gene expression of cyclin D1 and EGFR. These results provided a novel insight into the roles and mechanisms of curcumin in inhibition of colon cancer cell growth, and potential therapeutic strategies for treatment of colon cancer.



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Fig. 8. Schema of the mechanisms underlying curcumin inhibition of colon cancer Moser cell growth. Arrows indicate mechanisms from PPAR to cell growth and from curcumin to cell growth. ???, Mechanisms are not clear.

 

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This work was supported by a grant from the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (LSUHSC-S), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47995 (both to A. Chen), and starting funds from the Department of Pathology, LSUHSC-S.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Chen, Depts. of Pathology, Louisiana State Univ. Health Sciences Center in Shreveport, 1501 Kings Hwy, Shreveport, LA 71130 (E-mail: achen{at}lsuhsc.edu)

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.


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