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Symposium: Diet Induced Changes in the Colonic Environment and Colorectal Cancer

Ursodeoxycholic Acid (UDCA) Can Inhibit Deoxycholic Acid (DCA)-induced Apoptosis via Modulation of EGFR/Raf-1/ERK Signaling in Human Colon Cancer Cells^1,2

Arizona Cancer Center, Department of Radiation Oncology, University of Arizona, Tucson, AZ 85724

³To whom correspondence should be addressed. E-mail: jmartinez{at}azcc.arizona.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is known as a cytoprotective agent. UDCA prevents apoptosis induced by a variety of stress stimuli including cytotoxic bile acids such as deoxycholic acid (DCA). Here we examined the molecular mechanism by which UDCA can antagonize DCA-induced apoptosis in human colon cancer cells. UDCA pretreatment decreases the number of apoptotic cells caused by exposure to DCA and UDCA. Further studies of the signaling pathway showed that UDCA pretreatment suppressed DNA binding activity of activator protein-1 and this was accompanied by downregulation of both extracellular signal-regulated kinase (ERK) and Raf-1 kinase activities stimulated by exposure to DCA. DCA was also found to activate epidermal growth factor receptor (EGFR) activity and UDCA inhibited this. Collectively, these findings suggest that the inhibitory effect of UDCA in DCA-induced apoptosis is partly mediated by modulation of EGFR/Raf-1/ERK signaling.

KEY WORDS: • apoptosis • colon cancer • deoxycholic acid • ursodeoxycholic acid

Bile acids have long been implicated in the process of colon cancer development and cholestatic liver disease (1). Different bile acids have been shown to exhibit different biological activities (2) and previous studies suggest that this may be related to their chemical structure (3). For example, highly hydrophobic bile acids, such as chenodeoxycholic acid (CDCA)⁴ and deoxycholic acid (DCA), are able to induce apoptosis, whereas hydrophilic bile acids, such as ursodeoxycholic acid (UDCA), are not cytotoxic in colorectal cancer cells (4). In fact, UDCA inhibits DCA-induced apoptosis production in rat hepatocytes and nonhepatic cells in vitro by modulating mitochondrial membrane perturbation, reducing Bax protein abundance in mitochondria, as well as inhibiting reactive oxygen species (5,6).

Despite numerous studies the molecular mechanism by which UDCA can exert protective effect on the damaged cells is not clear yet. Intensive investigation has demonstrated that bile acids activate cytoplasmic protein kinase cascades and contribute to inducing some proto-oncogenes, such as activator protein-1 (AP-1) and cyclooxygenase-2 (7). Moreover, previous studies from our lab and others showed that bile acids stimulated intracellular signaling pathway including protein kinase C (PKC) and mitogen-activated protein kinases and this combined to activate AP-1 (8). Increasing evidence supports the idea that DCA-induced signaling pathway is mediated through the activation of receptor tyrosine phosphorylation kinase, epidermal growth factor receptor (EGFR) and downstream activation of Raf-1/MAP-kinase/ERK-kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade (9). Here we show that UDCA exerts an anti-apoptotic effect by inactivation of intracellular signaling which has been stimulated by DCA and that this anti-apoptotic activity is mediated in part through suppression of EGFR/Raf-1/ERK signaling.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Reagents

DCA was obtained from Sigma Chemical and UDCA was obtained from Calbiochem. All of the bile acids were maintained as 100 mmol/L stock solutions in water and were stored at 4°C. The stock solution was stored at -20°C. ZD1839 (Iressa) was provided by AstraZeneca and was maintained as a stock solution in DMSO.

Cell culture and treatment

HCT116 (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 100 U/ml penicillin/streptomycin at 37°C in an incubator containing 5% CO₂. For experiments, cells were grown to 80–95% confluency. Bile acids were diluted in culture medium before the experiments.

Apoptosis assay

Apoptosis was quantified as described previously (10).

Western blot analysis

Total cellular protein was extracted as described (11) and subjected to SDS-polyacrylamide gel electrophoresis in 12.5% gel. Western blot analyses were performed using rabbit polyclonal antibodies against p-ERK (Cell signaling) proteins and rabbit polyclonal antibodies against human -tubulin (Santa Cruz). Bound antibody was visualized using chemiluminescent substrate (SuperSignal West Pico) and exposed to X-ray film (Kodak).

Isolation of nuclear protein and gel shift assay

Cells were homogenized with a Dounce homogenizer in lysis buffer [10 mmol/L HEPES, pH 7.9 (at 4°C), 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.2 mmol/L phenylmethysulfonyl fluoride, and 0.5 mmol/L dithiothreitol]. Nuclear protein was isolated and gel shift assays were performed as described previously (11).

Raf-1 Immunoprecipitation kinase assay

Raf-1 kinase activity was measured by immumoprecipitation with an anti-Raf-1 antibody using a commercially available kit as recommended by the manufacturer (Upstate Biotechnology).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
UDCA suppresses DCA-induced apoptosis

DCA induced apoptosis in HCT116 human colon cancer cell line as previously reported (2); however UDCA did not exert cytotoxicity in HCT116 cells. Moreover, UDCA was protective in certain experimental conditions (5) in accord with reports concerning its chemopreventive role in colon tumorigenesis (12). To ascertain the effect of UDCA, we asked if UDCA protects cells from DCA-induced apoptosis. HCT116 cells were pretreated with 500 µmol/L UDCA for 18 h and then incubated with 500 µmol/L DCA for four h after removing UDCA (Fig. 1). Quantitation of apoptosis in UDCA pretreated cells showed that cell death was reduced by about 45% compared with cells that had been treated with DCA only. Hence, the UDCA pretreatment conferred resistance to DCA-induced apoptosis.

View larger version (28K): [in this window] [in a new window]   FIGURE 1 UDCA suppresses DCA-induced apoptosis. HCT116 cells were either untreated (control), treated with 500 µmol/L UDCA for 18 h (UDCA), treated with 500 µmol/L DCA for 4 h (DCA) or treated with 500 µmol/L UDCA for 18 h, the UDCA removed, and then exposed to 500 µmol/L DCA for 4 h (UDCA + DCA). Apoptosis was quantitated as described in Materials and Methods. Bars represent the average from three experiments. Error bars represent ± SD.

Our previous studies demonstrated that DCA induced AP-1 DNA binding and transactivation activities via activation of ERK (11). Therefore, we determined whether the protective effect of UDCA acts by modulating AP-1 DNA binding activity in DCA stimulated cells. Activation of AP-1 was first examined by gel shift assays. DCA causes an increase in AP-1 activity that peaks 4 h after treatment and this was suppressed, but not completely eliminated, by pretreating with UDCA. After 6 h treatment with DCA, AP-1 activity was abolished due to increase of apoptosis (Fig. 2A).

View larger version (33K): [in this window] [in a new window]   FIGURE 2 UDCA suppresses DCA-mediated activation of AP-1 and ERK. (A) HCT116 cells were either untreated, or pretreated with 500 µmol/L UDCA for 18 h and then UDCA was removed. Subsequently, the cultures were treated with 500 µmol/L DCA for different time periods as indicated and then cells harvested and AP-1 DNA binding activity assayed in mobility shift assays. (B) HCT116 cells were treated as described in A and harvested. Protein extracts were examined by immunoblotting for phospho-ERK by immunoblotting.

UDCA alters Raf-1 kinase activity induced by DCA

Because Raf-1 is an upstream activator of MAP-kinase (MAPK), we examined the role of Raf-1 in bile acid-induced signaling. HCT116 cells were treated with 500 µmol/L UDCA for 18 h and then different concentrations of DCA were added to cells for 4 h in the absence of UDCA. Raf-1 kinase cascade assays using inactive MEK1, MAPK/ERK, and MBP showed that DCA activated Raf-1 kinase activity in a concentration-dependent manner (Fig. 3). Phosphorylation of Raf-1 protein started to increase after 1 h treatment of DCA and lasted up to 4 h (data not shown). UDCA pretreatment for 18 h caused inhibition of Raf-1 activity and DCA did not overcome this blockade (Fig. 3). This result suggests that in colon cancer cells DCA exerted its biological effect through activation of Raf-1 kinase and UDCA can inhibit this activation.

View larger version (26K): [in this window] [in a new window]   FIGURE 3 UDCA blocks Raf-1 kinase activity stimulated by DCA. HCT116 cells were treated with 500 µmol/L UDCA for 18 h and then UDCA was removed from the cells. The cells were treated with 500 µmol/L DCA and then harvested. Cell lysates were immunoprecipitated with anti-Raf-1 antibody, washed, and Raf-1 kinase assays were performed using MEK and ERK as substrates as described in Materials and Methods. The phosphorylation of MBP by ERK was measured in scintillation counter. Each bar represents the mean ± SD of three independent experiments.

DCA caused ligand-independent activation of EGFR in primary rat hepatocytes suggesting that EGFR was involved in DCA-stimulated cell signaling pathway (9). Therefore, we next determined if EGFR, upstream effector of ERK, participates in mediating UDCA-induced activation of cell survival signaling. HCT116 cells were pretreated with 500 µmol/L UDCA for 18 h and then were treated with 500 µmol/L DCA for 15 min in the absence of 500 µmol/L UDCA. Total cellular extracts were prepared and Western blot analyses for activated phospho-EGFR and EGFR were performed. The results showed that UDCA decreased p-EGFR expression induced by exposure to DCA for 15 min and EGFR expression did not change either DCA or UDCA (Fig. 4A).

View larger version (41K): [in this window] [in a new window]   FIGURE 4 UDCA modulates DCA-induced activation of EGFR. (A) HCT116 cells were treated with 500 µmol/L UDCA for 18 h and then 500 µmol/L DCA was added for 15 min after elimination of UDCA. Total proteins were extracted and Western blot analyses were performed using phospho-specific antibody against EGFR or anti-EGFR antibody. The result is representative from several independent experiments. (B) The cells were preincubated with 5, 10, and 20 µmol/L ZD1839 for 2 h and then 500 µmol/L of DCA was added for 4 h. Apoptosis assays were performed under the fluorescence microscope after staining. Each bar represents the mean ± SD of three independent experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In this study, we demonstrated that the cytotoxic bile acid DCA can activate the EGFR and associated ras/mek/erk pathway. This leads to increased activity of AP-1, a heteromeric transcription factor composed of known proto-oncogenes that have been implicated in cell proliferation, differentiation, and tumor promotion (13). That DCA can activate the EGFR was unexpected since the structure of DCA bears no resemblance to the structure of epidermal growth factor. There are two potential explanations for this. One is that DCA stimulates the production and release of EGF and that this leads to upregulation of receptor activity. A second possibility is that DCA perturbs plasma membrane structure and that this leads to receptor activation. We favor the latter because it has been shown that DCA disrupts membrane organization in the rat colon (14) and because mutations in the membrane-associated domain of the Her2/neu receptor results in constitutive activity suggesting that the receptor/membrane interaction is functionally significant (15). In any case our results are consistent with other reports of ligand independent activation of EGFR (9) and could account, at least in part, for the reported capability of DCA to induce cell proliferation (16).

Interestingly, we found that the nontoxic bile acid UDCA suppressed DCA-induced activation of AP-1 as well as of raf, erk and even the EGFR. Hence, it appears that UDCA acts by interfering with the initial events (e.g., receptor activation) that leads to activation of raf/mek/erk signaling. It is unclear how UDCA accomplishes this. However, it is consistent with UDCA’s reported activity as an inhibitor of tumor development in the rat AOM animal model of colon carcinogenesis (17). Importantly, suppression of EGFR-mediated signaling by UDCA correlated with suppression of DCA-induced apoptosis suggesting that EGFR activation facilitated the apoptotic response. This notion is supported by our results with the EGFR inhibitor IRESSA, which also blunted DCA-induced apoptosis. However, given the relatively small effect that IRESSA had on DCA-induced apoptosis compared with the UDCA pretreatment, it seems likely that UDCA acts through other mechanisms in addition to suppression of EGFR.

Collectively our results suggest that DCA, classified as a tumor promoter, stimulates oncogene activity and proliferative signaling, whereas UDCA, a purported chemopreventive agent, can suppress proliferative signaling. Thus, UDCA’s activities as a chemopreventive agent may be mediated, at least in part, through a molecular mechanism that involves suppression of proliferative signaling.

	ACKNOWLEDGMENTS

 
The authors would like to thank Wenqing Qi for his excellent assistance and support of these studies.

	FOOTNOTES

 
¹ Presented at the Experimental Biology meeting, April 11–15 2003, San Diego, CA. The symposium was sponsored by the American Society for Nutritional Sciences and supported in part by an educational grant from the Group Danone and the Nutrition Science Research Group at the National Cancer Institute. The proceedings are published as a supplement to The Journal of Nutrition. This supplement is the responsibility of the guest editors to whom the Editor of The Journal of Nutrition has delegated supervision of both technical conformity to the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. The opinions expressed in this publication are those of the authors and are not attributable to the sponsors or the publisher, editor, or editorial board of The Journal of Nutrition. The Guest Editors for the symposium publication are Jon A. Story, Department of Foods and Nutrition, Purdue University, West Lafayette, IN, and J. Glenn Morris, Jr., Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, MD.

² This study was supported by National Institutes of Health Grant CA72008.

⁴ Abbreviations used: AP-1, activator protein-1; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MAPK, MAP-kinase; MEK, MAP-kinase/ERK-kinase; UDCA, ursodeoxycholic acid.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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7. Zhang, F., Subbaramaiah, K., Altorki, N. & Dannenberg, A. J. (1998) Dihydroxy bile acids activate the transcription of cyclooxygenase-2. J. Biol. Chem. 273:2424-2428.[Abstract/Free Full Text]

8. Rust, C., Karnitz, L. M., Paya, C. V., Moscat, J., Simari, R. D. & Gores, G. J. (2000) The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J. Biol. Chem. 275:20210-20216.[Abstract/Free Full Text]

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12. Neuman, M. G., Cameron, R. G., Shear, N. H., Bellentani, S. & Tiribelli, C. (1995) Effect of tauroursodeoxycholic and ursodeoxycholic acid on ethanol-induced cell injuries in the human Hep G2 cell line. Gastroenterology 109:555-563.[Medline]

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17. Earnest, D. L., Holubec, H., Wali, R. K., Jolley, C. S., Bissonette, M., Bhattacharyya, A. K., Roy, H., Khare, S. & Brasitus, T. A. (1994) Chemoprevention of azoxymethane-induced colonic carcinogenesis by supplemental dietary ursodeoxycholic acid. Cancer Res. 54:5071-5074.[Abstract/Free Full Text]

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