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Mol Cancer Ther. 2006;5:68-79
© 2006 American Association for Cancer Research

Enhancement of DNA topoisomerase I inhibitor–induced apoptosis by ursodeoxycholic acid

Tadashi Ikegami1,2, Yasushi Matsuzaki1, Maryam Al Rashid4, Susan Ceryak2,3, Yining Zhang1 and Bernard Bouscarel2,4

1 Division of Gastroenterology and Hepatology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba City, Japan and 2 Departments of Medicine, 3 Pharmacology and Physiology, and 4 Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, District of Columbia

Requests for reprints: Bernard Bouscarel, George Washington University Medical Center, 2300 Eye Street, Northwest Ross Hall, Room 523, Washington, DC 20037. Phone: 202-994-2114; Fax: 202-994-3435. E-mail: bbouscarel{at}mfa.gwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Certain hydrophobic bile acids, including deoxycholic acid and chenodeoxycholic acid, exert toxic effects not only in the liver but also in the intestine. Moreover, ursodeoxycholic acid (UDCA), which has protective actions against apoptosis in the liver, may have both protective and toxic effects in the intestine. The goal of the present study was to clarify the mechanisms responsible for the toxic effect of UDCA in intestinal HT-29 cells. Here, we show that UDCA potentiated both phosphatidylserine externalization and internucleosomal DNA fragmentation induced by SN-38, the most potent metabolite of the DNA topoisomerase I inhibitor, CPT-11. Furthermore, the loss of mitochondrial membrane potential as well as mitochondrial membrane permeability transition induced by SN-38 was enhanced in the presence of UDCA, resulting in an increased lethality determined by colony-forming assay. This UDCA-induced increased apoptosis was not due to alteration of either intracellular accumulation of SN-38 or cell cycle arrest by SN-38. The increased apoptosis was best observed when UDCA was present after SN-38 stimulation and was independent of caspase-8 but dependent on caspase-9 and caspase-3 activation. Furthermore, UDCA enhanced SN-38-induced c-Jun NH2-terminal kinase activation. In conclusion, UDCA increases the apoptotic effects while decreasing the necrotic effects of SN-38 when added after the topoisomerase I inhibitor, showing potential clinical relevance as far as targeted cell death and improved wound healing are concerned. However, the use of this bile acid as an enhancer in antitumor chemotherapy should be further evaluated clinically. [Mol Cancer Ther 2006;5(1):68–79]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CPT-11 [7-ethyl-10-4-(1-piperidino)-1-piperidinocarbonyloxycamptothecin] has been approved worldwide for the treatment of colorectal cancer and is under extensive investigation and therapeutic evaluation for a variety of other cancers (1–4). Both CPT-11 and SN-38, its 7-ethyl-10-hydroxycamptothecin derivative from carboxylesterase-induced hydrolysis (5), present antitumor activity through the inhibition of DNA topoisomerase I (6). SN-38 has at least a 1,000-fold more potent antitumor effect than CPT-11 as shown in vitro (6). CPT-11 and SN-38 stabilize the topoisomerase I-DNA complex, and its collision with the DNA replication fork leads to the generation of permanent strand breaks and to cell death (7). Stabilization of the cleavable complexes by CPT-11/SN-38 is accompanied by a G2-M arrest and apoptosis (8, 9). Although the mechanism is still unclear, the apoptotic effect of CPT-11 associated with an increased DNA fragmentation has been reported previously in a variety of colon cancer cell lines (8). The apoptotic effect of CPT-11 in colon carcinoma and/or lung cancer cells has been linked not only with an increased cleavage of poly(ADP-ribose) polymerase, characteristic of programmed cell death, but also with an alteration of proapoptotic and antiapoptotic proteins, including Bax, Bcl-xL, and Bcl-2 (8, 10). Furthermore, these apoptotic effects of the camptothecin derivatives are at least p53 dependent (see ref. 11 for review).

Bile acids are believed to play an important role in the etiology of colorectal cancer, which is one of the leading causes of cancer-related deaths in the world (12). Several studies consider hydrophobic secondary bile acids, such as deoxycholic acid, to be tumor promoters and to exert their cytotoxicity through apoptosis (13, 14). One of the possible apoptotic effects of bile acids is attributed to the perturbation of mitochondrial functions (15, 16). As a result of damage and loss of transmembrane potential, the mitochondria either release cytochrome c or Smac/DIABLO into the cytosol. The former, in turn, binds to Apaf-1 and induces caspase-9-dependent activation of caspase-3, whereas the latter blocks the inhibitory effects of inhibitor of apoptosis proteins on caspases (17, 18). Both pathways promote cell death.

Not all bile acids have cytotoxic properties. The dihydroxy bile acid, ursodeoxycholic acid (UDCA), has been shown to exert antiapoptotic effects (15). Although the antiapoptotic mechanism of UDCA is still under discussion, several reports have hypothesized this bile acid to stabilize the mitochondrial structure (15). However, this bile acid may also have proapoptotic actions: (a) UDCA potentiated photodamage in leukemia cells (19), (b) UDCA did not protect against apoptosis induced by hydrophobic bile acids in several colonic cancer cell lines (20), and (c) UDCA induced apoptosis in hepatocytes when both mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase pathways were inhibited (21). Therefore, UDCA may act differentially on death and survival pathways depending on the cell type, physiologic conditions, and/or stimulus. Thus, the clarification of the mechanism of UDCA action especially in terms of antiapoptotic or proapoptotic effects is relevant to foster a better understanding and to widen the clinical usage of this bile acid. The hypothesis that UDCA potentiates the SN-38-induced cytotoxicity, which in turn could increase the chemotherapeutic effect of this camptothecin derivative, is attractive. Indeed, a recent report indicated that coadministration of UDCA with photodynamic therapy resulted in augmented antitumor effects (19).

The aim of the present study was to investigate the mechanism of action of UDCA on the series of events associated with cell death, such as cell cycle alteration, apoptosis, and growth inhibition induced by SN-38. The possible involvement of mitochondrial membrane potential ({Delta}{Psi}m) and caspases in the action of UDCA was also examined. An attempt was made to delineate key protein kinases activated by this bile acid. The colon-derived adenocarcinoma HT-29 cell line, a representative colon cancer cell line, was used as the predominant model for these studies. However, the colonic adenocarcinoma LS174T and Caco-2 and the hepatic HepG2 cells were used for comparison.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
SN-38 and radiolabeled SN-38 ([14C]SN-38) were kindly supplied by Yakult Honsha Co. Ltd. (Tokyo, Japan) and were 98% to 99% pure as judged by gas-liquid chromatography. UDCA, tauroursodeoxycholic acid, chenodeoxycholic acid, and taurocholic acid were kindly supplied by Mitsubishi Welpharma Co. Ltd. (Osaka, Japan). Green fluorescent protein (GFP)–labeled Annexin V was obtained from Clontech (Palo Alto, CA). Propidium iodide (PI) was obtained from Sigma Chemical Co. (St. Louis, MO). Tetramethylrhodamine, xanthylium, 3,6-bis(dimethylamino)-9-[2-(methoxycarbonyl) phenyl], perchlorate (TMRM; Mitotracker-1) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were obtained from Molecular Probes (Eugene, OR). The specific caspase inhibitors (z-DEVD-fmk, z-IETD-fmk, z-LEHD-fmk, and Z-VAD-fmk) were obtained from Trevigen, Inc. (Gaithersburg, MD). PD98059 and SP600125 were obtained from Calbiochem (San Diego, CA). The human colon adenocarcinoma cell line HT-29 was from the American Type Culture Collection (Manassas, VA). DMEM was from Life Technologies, Inc. (Frederick, MD).

Cell Culture and Clonogenic Assay
HT-29 cells were grown in DMEM containing 10% fetal bovine serum, 50 units/mL penicillin G, and 50 µg/mL streptomycin at pH 7.4 and maintained at 37°C in a humidified atmosphere of 5% CO2. Clonogenic lethality of SN-38 and UDCA was determined as described previously (9). In brief, the HT-29 cells were treated with increasing concentrations of SN-38 for 2 hours. After removal of the drug by washing twice with PBS, the cells were further incubated for 24 hours in the presence or absence of UDCA. Cells (n = 500) were reseeded in triplicate in 60-mm culture dishes containing 3 mL DMEM. The colonies were grown for 2 weeks, washed with PBS, fixed with 80% methanol, stained with methylene blue (0.04%), and counted using an Eagle Eye II transilluminator (Stratagene, La Jolla, CA). During colony growth, the culture medium was replaced every 3 days. Cloning efficiency for untreated HT-29 cells was ~74%.

Semiquantification of DNA Fragmentation
The comet assay or single-cell gel electrophoresis assay is based on the alkaline lysis of labile DNA at sites of damage. The unwound, relaxed DNA is able to migrate out of the cell during electrophoresis and can be visualized by SYBR Green (Molecular Probes) staining. Cells that have accumulated DNA damage appear as fluorescent comets with tails of DNA fragmentation or unwinding, whereas normal undamaged DNA does not migrate far from the origin. The fluorescence intensity of these cells was determined by using an ACAS570 laser scanner cytometer (Meridian Instruments, Inc., Okemos, MI). The tail length and the head were selected for the quantification of DNA migration, and the ratio of these values was calculated.

Annexin V and PI Staining
The apoptotic HT-29 cells were determined by using GFP-labeled Annexin V and in accordance with the manufacturer's instructions as described previously (9). Binding affinity for GFP-Annexin V and PI was determined by flow cytometric analysis (FACScan, Becton Dickinson, San Jose, CA). Excitation wavelength was 488 nm, and the emission wavelengths were 530 nm (FL1) for GFP and 620 nm (FL2) for PI. Fluorescence cutoff for the FL1 and FL2 channel was defined using HT-29 cells permeabilized with 0.1% Triton X-100–containing PBS.

Effect of SN-38 and UDCA on mRNA Expression Level of Key Proteins Regulating Cell Cycle and Apoptosis
mRNA expression level was quantitated by RNase protection assay with a 32P-labeled multitranscript probe containing gene sequences for the antiapoptotic proteins Bcl-W, Bcl-xL, and Bcl-2, the proapoptotic proteins Bcl-xS and Bax, and the cyclin-dependent kinase inhibitors, p21waf1/cip, p15INK4B, and p16INK4A according to the manufacturer's instructions (BD PharMingen, San Diego, CA). Protected 32P-labeled probes were resolved on a 5% acrylamide sequencing gel, and the dried gel was exposed to a phosphor screen (Amersham, Piscataway, NJ). Relative expression was determined by densitometric analysis, using a Molecular Dynamics STORM PhosphorImager (Piscataway, NJ) and ImageQuant software, and normalized to the respective expression of two housekeeping genes, L32 and glyceraldehyde-3-phosphate dehydrogenase. Finally, the normalized results were expressed as percentage of the respective control.

Determination of {Delta}{Psi}m
HT-29 cells were incubated with 1 µg/mL of the {Delta}{Psi}m-sensitive dye, JC-1, for 30 minutes at 37°C, gently harvested with trypsin, washed in PBS, resuspended in medium at a density of ~1 x 107/mL, and transferred on ice to the flow cytometer. JC-1 was excited at 488 nm and the monomer signal (green) was analyzed at 525 nm (FL1) on a flow cytometer using a minimum of 10,000 cells per sample. Simultaneously, the aggregate signal (red) was analyzed at 590 nm (FL2). FCCP (1 µmol/L, Sigma), a known mitochondrial inhibitor, was used as positive control for mitochondrial depolarization. The ratio of the signal at 590 nm to the signal at 525 nm was determined after background subtraction and expressed as percentage of control.

Confocal Laser Scanning Microscopy
To visualize the mitochondria permeability transition, the HT-29 cells were stained with TMRM and acetoxymethylester of calcein (calcein-AM, Wako Chemicals, Tokyo, Japan) and observed by confocal laser scanning microscopy (Leica TCS SP2, Leica Microsystems AG, Wetzlar, Germany) as reported previously by Nieminen et al. (22). In brief, HT-29 cells were loaded in culture medium with 500 nmol/L TMRM and 1 µmol/L calcein-AM for 20 minutes at 37°C, washed with PBS, and mounted on the microscope stage. Under this condition, calcein-AM accumulated virtually exclusively in the cytosol, and mitochondria were imaged as voids in the cytosolic fluorescence. Onset of permeability of mitochondrial membranes to calcein-AM was indicated by filling of mitochondrial voids.

Topoisomerase I, c-Jun NH2-Terminal Kinase, and Extracellular Signal-Regulated Kinase Expression Levels and Caspase Activity Determination
Total cellular proteins (10–20 mg/mL) were separated by SDS-PAGE according to the method of Laemmli (23) and transferred to polyvinylidene difluoride membranes. Blots were probed with specific antibodies to the respective ß-actin and topoisomerase I (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated and total c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK; Cell Signaling, Beverly, MA) followed by the appropriate secondary horseradish peroxidase–labeled antibody. The immunoreactive proteins were visualized by enhanced chemiluminescence, analyzed by densitometric scanning, and normalized to the respective ß-actin or total kinase absorbance signals.

The specific caspase activity in HT-29 cell lysates was determined using a caspase colorimetric activity assay kit (Chemicon International, Temecula, CA) according to the manufacturer's instruction.

Statistical Analysis
Except as otherwise indicated, results were expressed as mean ± SE. The statistical significance of the means was determined by either one-way ANOVA or Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
UDCA Enhanced the Lethality of SN-38-Treated HT-29 Cells
Exposure of HT-29 cells to 50 µmol/L UDCA alone for 24 hours had no effect on colony-forming ability (data not shown). The colony-forming ability of the cells treated with 0.5 µmol/L SN-38 for 2 hours was significantly (P < 0.001) inhibited by ~50% when compared with control (Fig. 1A ). After removal of SN-38, the addition of 2.5 to 10 µmol/L UDCA was without effect; however, the addition of 50 µmol/L UDCA for 24 hours resulted in a significant (P < 0.01) further reduction in colony formation by 20% to 25% when compared with that of the cells treated with SN-38 alone, supporting an UDCA-induced enhancement of SN-38 clonogenic lethality. Furthermore, SN-38 concentrations ≥0.05 µmol/L significantly increased clonogenic lethality by 20% to 50%, whereas in the presence of 50 µmol/L UDCA a SN-38-induced clonogenic lethality of 30% was observed at a concentration as low as 0.005 µmol/L (Fig. 1B). Moreover, although still under investigation, the UDCA-induced increase in SN-38 lethality was apparently independent of an alteration in topoisomerase I protein expression level (data not shown).


Figure 1
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  		Figure 1. Growth inhibition of HT-29 cells and topoisomerase I protein expression induced by SN-38 and UDCA. A, HT-29 cells were incubated for 2 h with 0.5 µmol/L SN-38. After removal of SN-38, the cells were further incubated with increasing concentrations of 2.5 to 50 µmol/L UDCA for 24 h, collected by trypsinization, and then seeded in fresh culture dishes (500 per dish). Colonies grown after 2 wks were counted and expressed as percentage of control (CTL). B, HT-29 cells were incubated for 2 h with increasing concentrations (0.005–0.5 µmol/L) of SN-38, and after removal, the cells were further incubated with 50 µmol/L UDCA for 24 h and processed as described above. Points, mean of three independent experiments; bars, SE. *, P < 0.01, significantly different from SN-38 treatment without UDCA.

 
Effect of UDCA on SN-38-Induced Cell Death
We have reported previously that 35% of the cells treated with 0.5 to 1 µmol/L SN-38 had externalized phosphatidylserine on the plasma membrane, one of the early-phase phenomena of apoptotic cell death (9). Thus, we studied whether the magnitude of the phosphatidylserine externalization induced by SN-38 was altered by UDCA. In this assay, both Annexin V– and PI-positive cells were considered to be necrotic as described previously (9). For these studies, both floating and attached cells were collected and subjected to FACS analysis. As shown in Fig. 2A , 0.5 µmol/L SN-38 alone induced phosphatidylserine externalization in 16 ± 4.3% of the cells after the 48-hour incubation period. Under these conditions, the percentage of necrotic cells (Annexin V– and PI-positive) was 33.2 ± 4.6%. The addition of UDCA after initial SN-38 exposure presented a dose-dependent increase in Annexin V–positive cells (early apoptosis). This increase reached ~60% in the presence of 100 µmol/L UDCA and was significantly different from control (P < 0.001). Concurrently, the percentage of Annexin V/PI–positive cells (primary and secondary necrosis) was significantly decreased in a dose-dependent manner to 23.4 ± 1.2% with 100 µmol/L UDCA. However, UDCA alone had no significant effect on phosphatidylserine externalization at the various concentrations tested. Furthermore, although not shown, the majority of the Annexin V–positive cells were among the floating rather than attached cells. This suggests that, in our system, the cell detachment occurred at an early phase in SN-38-induced HT-29 cell death. To determine whether this effect was cell specific, we did similar studies using colonic adenocarcinoma Caco-2 and LS174T cells as well as the hepatocarcinoma HepG2 cell line. The cells were incubated with 1 µmol/L SN-38 for 2 hours and, after washing, for an additional 72 hours without or with 100 µmol/L UDCA. Apoptosis was determined as described above. The results in Fig. 2B indicate that the significantly increased apoptotic effect of UDCA in the presence of SN-38 was not specific to HT-29 cells and was also observed in certain other colonic (LS174T) and hepatic (HepG2) cell lines. However, although the tendency remained, the effect of UDCA on Caco-2 cells was not significant.


Figure 2
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  		Figure 2. SN-38-induced phosphatidylserine externalization is enhanced by UDCA. A, HT-29 cells were incubated in the presence and absence of 0.5 µmol/L SN-38 for 2 h. After replacement of the culture medium, the cells were further incubated with increasing concentrations (1–100 µmol/L) of UDCA for 48 h. Percentage of apoptotic and necrotic cells was determined by flow cytometry using both GFP-Annexin V and PI. The GFP only–positive cells were defined as apoptotic, whereas GFP- and PI-positive cells were defined as necrotic. Points, mean percentage of control from two independent experiments done in duplicate; bars, SE. *, P < 0.001, significantly different from control. B, under the same conditions, HT-29, LS174T, Caco-2, and HepG2 cells were incubated in parallel with 1 µmol/L SN-38 in the presence (+) or absence (-) of 100 µmol/L UDCA for 72 h. Apoptotic cells were determined by Annexin V/PI binding assay. *, P < 0.01; **, P < 0.05.

 
Optimal Incubation Period for the Proapoptotic Action of UDCA
Preincubation with UDCA is prerequisite for its proapoptotic action on a photoirradiated cancer cell line (19). Therefore, to clarify the critical period of incubation of the cells with UDCA to induce optimum SN-38 toxicity, the HT-29 cells were incubated with 100 µmol/L UDCA either 24 hours before SN-38 exposure (pre-UDCA), during the 2-hour SN-38 exposure (co-UDCA), or after SN-38 exposure (post-UDCA) and for an additional 72 hours (Fig. 3A ). In addition, in a subsequent experiment, UDCA was added after SN-38 removal and for either 72 hours or the last 48 and 24 hours of the experiment (Fig. 3B). The phosphatidylserine externalization was then quantified as indicated in Materials and Methods. Although UDCA addition had no effect when added before or during SN-38 exposure (Fig. 3A), it increased SN-38-induced apoptosis by >100% when added after removal of SN-38. The increased UDCA effect was significant after a period as short as 24 hours (30%) and was a function of the period of incubation with this bile acid (Fig. 3B).


Figure 3
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  		Figure 3. Effect of the period of incubation of UDCA on SN-38-induced apoptosis. A, HT-29 cells were incubated with 0.5 µmol/L SN-38 for 2 h and in the absence (w/o UDCA) and presence of 100 µmol/L UDCA for various periods. Pre UDCA, cells were incubated with UDCA for 24 h, removed, and then incubated with SN-38 for 2 h; Co UDCA, cells were incubated with SN-38 and UDCA simultaneously for 2 h; Post UDCA, after 2-h incubation with SN-38, cells were further incubated with UDCA for 72 h. *, P < 0.01, significantly different compared with w/o UDCA. B, cells were incubated in the absence (CTL) or presence of SN-38 for 2 h and then washed with PBS and further incubated with UDCA (100 µmol/L) for the last either 24, 48, or 72 h. Percentage of apoptotic and/or necrotic cells at the end of the incubation period was determined by flow cytometric analysis using Annexin V/PI. *, P < 0.05, significantly different from SN-38 alone.

 
Apoptotic Action of SN-38 and UDCA Detected by DNA Fragmentation
DNA fragmentation represents a hallmark of apoptosis regardless of whether the apoptotic process has been initiated by either intrinsic signals or death receptors involving extrinsic signals. Two major steps have been identified in the apoptosis-associated DNA fragmentation. The first step involves formation of high molecular weight DNA fragments of 50 to 300 kb. The second step generates small, 200- to 300-bp DNA fragments. These small fragments lead to DNA ladder formation classically associated with apoptosis. Initially, we determined the effect of UDCA on SN-38-induced DNA fragmentation by using DNA electrophoresis. UDCA alone has no effect on DNA fragmentation. However, the electrophoresis of the small molecular weight DNA revealed that 0.5 to 2 µmol/L SN-38 alone induced DNA fragmentation in a dose-dependent fashion (Fig. 4A ). Furthermore, the addition of 50 µmol/L UDCA after removal of SN-38 and for 72 hours enhanced this DNA ladder formation induced by SN-38 at every concentration tested.


Figure 4
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  		Figure 4. Induction of DNA fragmentation by SN-38 and UDCA. A, HT-29 cells were incubated with SN-38 for 2 h, washed with PBS thrice, and then further incubated in the presence or absence of 50 µmol/L UDCA for 72 h. Cells were collected and electrophoresed in 0.5% agarose gels. B, cells were incubated with 1 µmol/L SN-38 for 2 h and with 100 µmol/L UDCA for either 24 or 48 h after removal of SN-38. The cells loaded on low melting point agarose gels were permeabilized after solidification of the gel. Fragmented nucleic DNA migrates according to the molecular size. C, fluorescence intensity of these comets was visualized with a Meridian laser spectrometer, and the ratio of the tail over the head of the comet was calculated as described in Materials and Methods. Representative of three independent experiments. *, P < 0.005; **, P < 0.05, significantly different from respective control.

 
For quantification purposes and to support these above observations, single-cell electrophoresis (i.e., comet assay) was used. In this assay, fragmented DNA can migrate through permeabilized nuclear membranes as well as plasma membranes, producing a "tail" from the nuclear head. The overall shape resembles that of a comet. Either control cells or 100 µmol/L UDCA-treated cells did not induce any significant DNA tail formation (Fig. 4B and C). SN-38 treatment induced the formation of a tail suggesting DNA fragmentation in these cells, mimicking the smear pattern observed in conventional agarose gel electrophoresis (Fig. 4A). Furthermore, the addition of UDCA resulted in the extension of the tail induced by SN-38 alone, which represents greater DNA fragmentation (Fig. 4B). The calculated ratio of the fluorescence intensity of the tail to head increased in a time-dependent fashion by SN-38 (Fig. 4C). Although the tail formation in the cells treated with SN-38 alone was not significantly different from control, the addition of 100 µmol/L UDCA for 24 hours induced a significant ~3-fold increase (P < 0.01). At 48 hours, SN-38 induced a 15.2-fold increase in tail formation compared with control (P < 0.01), and the addition of 100 µmol/L UDCA further enhanced the tail formation by ~1.6-fold (P < 0.05), suggesting the facilitation of apoptotic cell death by UDCA. Taken together, these results support the enhancement of SN-38 lethality by UDCA to be due to the augmentation of apoptotic cell death.

Effect of UDCA on HT-29 Cellular Uptake and Efflux of SN-38 and on Cell Cycle
The intracellular accumulation of SN-38 in the presence of UDCA was determined to assess any possible alteration in the HT-29 cellular uptake or efflux of SN-38 by the bile acid. After 2-hour incubation, SN-38 was removed and the cells were further incubated with 100 µmol/L UDCA. The intracellular accumulation of SN-38 in HT-29 cells was measured with [14C]SN-38. The intracellular concentrations following 2- and 24-hour incubation of the cells with 0.5 µmol/L SN-38 were 70 ± 11 and 59 ± 4 pmol/106 cells and were not significantly different (68 ± 7 and 70 ± 5 pmol/106 cells) when determined in the presence of 100 µmol/L UDCA. Under the same conditions, the intracellular concentrations following 2- and 24-hour incubation of the cells with 1 µmol/L SN-38 were 109 ± 5 and 158 ± 32 pmol/106 cells and again were not significantly different (123 ± 32 and 144 ± 18 pmol/106 cells) when tested in the presence of 100 µmol/L UDCA. These results are the mean ± SE of four to six different experiments.

Furthermore, SN-38 was shown to alter the HT-29 cell cycle characterized by a dose-dependent decrease in the percentage of cells in G0-G1 phase, a parallel increase of the cells in S phase, and an increase of those in G2-M phase. A marked cell cycle arrest in S phase was observed with concentrations of SN-38 as low as 0.1 µmol/L but was not affected by the presence of 100 µmol/L UDCA for 24 hours (data not shown).

Effect of SN-38 and UDCA on the mRNA Expression Level of Proteins Associated to the Regulation of Both Apoptosis and Cell Cycle
One of the mechanisms of action of drug-induced cell death includes modulation of the expression of proteins associated to either apoptosis or cell cycle pathway. Therefore, we studied the effect of SN-38 either transiently present for 2 hours or after further incubation for 72 hours in the presence and absence of 50 µmol/L UDCA on the mRNA expression level of various proteins associated to these respective pathways. SN-38 reduced the mRNA level of Bcl-xL, p16, and c-fos by 30% to 50% but did not affect the mRNA expression level of Bax, Bcl-2, p21, and p15. The HT-29 cells have a mutated p53; therefore, this was used as an additional internal control. However, in these experiments, it was clear that UDCA had no significant effect either alone or when added after removal of SN-38 on the mRNA level of these proteins (data not shown). These results on the lack of effect of UDCA on transcriptional regulation of proteins of the cell cycle are supportive of the above report that UDCA did not alter the SN-38-induced cell cycle arrest.

Effect of UDCA on {Delta}{Psi}m
The involvement of the mitochondria in the apoptotic effect of SN-38 and UDCA was investigated. When the mitochondrial membrane is hyperpolarized, JC-1 forms J-aggregates in the mitochondria, which emit a red orange fluorescence detectable at 490-nm wavelength. However, when the mitochondrial membrane is depolarized, JC-1 emits a green fluorescence detectable at 525 nm. First, we used 1 µmol/L FCCP, a proton ionophore that is well known to depolarize {Delta}{Psi}m. Five to 10 minutes after application of FCCP, the population of cells detected at 525 nm increased, whereas that at 490 nm decreased, indicating that the mitochondrial membrane was depolarized (data not shown). Next, the effect of 1 µmol/L SN-38 and 100 µmol/L UDCA on the {Delta}{Psi}m over a period of 48 hours was investigated. Up to 6 hours, the number of cells with depolarized mitochondria was not altered by either SN-38 or UDCA alone. After 24 hours, this number was not significantly increased by SN-38 alone, whereas SN-38 followed by 24-hour incubation with 100 µmol/L UDCA induced a significant (P < 0.01) increase in cells with depolarized mitochondria by ~250% when compared with control (Fig. 5A ). By 48 hours, the percentage of cells with depolarized mitochondria was similarly increased by SN-38 alone (~300%) or by SN-38 followed by UDCA for 48 hours (~330%).


Figure 5
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  		Figure 5. Reduction of {Delta}{Psi}m and mitochondrial membrane permeability transition by SN-38 and UDCA. A, {Delta}{Psi}m was determined by flow cytometry using the fluorescent marker JC-1. HT-29 cells were treated with 0.5 µmol/L SN-38 for 2 h, washed with PBS, and then incubated up to 48 h in the presence (SN/UD) and absence (SN38) of 100 µmol/L UDCA. After trypsinization, JC-1 was loaded into HT-29 cells and analyzed by flow cytometry. Depolarized mitochondria formed aggregates and this aggregation resulted in the alteration in fluorescent emission wavelength (525–590 nm). The number of cells with depolarized mitochondria was counted and expressed as percentage of control. B, HT-29 cells were treated as described above. For the final 20 min of incubation, the cells were coloaded with calcein-AM (1 µmol/L) and TMRM (0.5 µmol/L). Representative images of the HT-29 cells loaded with either calcein-AM or TMRM before and after SN-38 treatment. C, HT-29 cells costained with calcein-AM and TMRM were scanned under confocal laser scanning microscope and a three-dimensional image was reconstructed from the images of serial scanning. CTL, untreated control cells; SN-38, HT-29 cells 48 h after incubation with 0.5 µmol/L SN-38 for 2 h. D, HT-29 cells were incubated, loaded, and scanned as described above. SN+UDC, SN-38 + UDCA condition; 1–6, serial confocal images collected as the focal plane was advanced in 0.2 µmol/L increments from the bottom of the cell layer. E, quantification of the rounded cells in SN-38-treated HT-29 cells. The number of rounded HT-29 cells was counted and expressed as percentage of total. In each group, total number of counted cells was 150 to 200. All the images were collected using a x63 objective oil immersion lens in an inverted microscope. Columns, mean of two independent experiments done in duplicate; bars, SE. *, P < 0.005, significantly different from control; **, P < 0.05, significantly different from SN-38-treated cells.

 
This was also confirmed by the visualization of depolarized mitochondria using confocal laser scanning microscopy with TMRM and calcein-AM according to the method developed by Nieminen et al. (22). Figure 5B and C reports a change in shape of the cells following SN-38 treatment for 48 hours. The HT-29 cells were rounder, larger, and positioned at a higher Z-axis from the bottom plane and were termed round cells. This was shown in a reconstructed three-dimensional image (Fig. 5C). This SN-38-induced increase in cell volume was cell density independent (data not shown). The polarized mitochondria were originally localized in the perinuclear area of the cells represented by the void space when loaded with calcein-AM as indicated by an arrow in Fig. 5B. However, the mitochondria were rather diffuse and obscured in the round cells, suggesting increased mitochondrial membrane permeability or damage in these cells (Fig. 5B and D). In the presence of UDCA for 48 hours, the number of round cells significantly increased (P < 0.005) when compared with those treated with SN-38 alone, and an abundant number of apoptotic bodies were detected (see arrow in SN-38 + UDCA; Fig. 5D). UDCA alone did not induce any significant mitochondrial or cell volume changes (data not shown). The percentage of the round cells after 48-hour incubation was quantified in Fig. 5E. After 48 hours, SN-38 increased the number of round cells from ~3% to ~19%. Furthermore, the addition of 100 µmol/L UDCA resulted in an increased number of round cells to ~48% (Fig. 5E). The number of round cells determined 24 hours following SN-38 treatment was not significantly different from that of control, whereas the addition of UDCA for 24 hours after SN-38 removal resulted in a significantly increased number of round cells (data not shown). These results are consistent with the reduction of {Delta}{Psi}m by the combination of SN-38 and UDCA (Fig. 5A), suggesting a further alteration of the mitochondrial membrane permeability transition by the combination of SN-38 and UDCA. As a positive control for mitochondrial membrane depolarization, we treated the cells with increasing concentrations of FCCP, an inhibitor of the mitochondrial oxidative chain. There was no increase in round cells, whereas the cells showed a diffuse pattern of depolarized mitochondria (data not shown). Therefore, the semidetachment of the cells is a phenomenon induced by SN-38 independently of its damaging effect on mitochondria alone.

Role of Caspase-3, Caspase-8, and Caspase-9 on the Combined Apoptotic Effect of SN-38 and UDCA
To test the hypothesis that UDCA activates the mitochondrial apoptotic pathway, we determined the caspase activities in treated HT-29 cells. As shown in Fig. 6A , SN-38 induced a significant activation of caspase-3 and caspase-8. The caspase-9 activity was slightly elevated by SN-38 alone but was not statistically significant. The addition of 100 µmol/L UDCA for 24 hours resulted in the further significant increase of both caspase-3 and caspase-9 activities without affecting that of caspase-8 (Fig. 6A). Furthermore, the involvement of caspases in the UDCA-induced increased apoptotic effect of SN-38 was investigated using specific caspase inhibitors. The increased effect of UDCA on both SN-38-induced apoptosis and formation of round cells was almost completely abolished with 20 µmol/L of the general caspase inhibitor, ZVAD-fmk (Fig. 6B). The percentage of apoptotic cells induced by SN-38 followed with UDCA treatment for 24 hours was not significantly decreased in the presence of a specific caspase-8 inhibitor (IETD), whereas the incubation with either caspase-3 (DEVD) or caspase-9 (LEHD) inhibitor resulted in ~45% inhibition of the UDCA effect (Fig. 6B).


Figure 6
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  		Figure 6. Role of caspases in the apoptotic effect of SN-38 + UDCA. A, HT-29 cells were incubated with 0.5 µmol/L SN-38 (SN) for 2 h. After removal of SN-38, the cells were further incubated with 100 µmol/L UDCA (UD). Caspase activity in the cell lysate of the treated cells was determined by colorimetric assay as described in Materials and Methods. Columns, mean percentage of control determined in the absence of both SN-38 and UDCA from two independent experiments done in duplicate; bars, SE. *, P < 0.01, significantly different from control; **, P < 0.05, significantly different from the condition with SN-38 alone. B, HT-29 cells were incubated with 0.5 µmol/L SN-38 for 2 h. After removal of SN-38, the cells were further incubated with 100 µmol/L UDCA in the presence of 20 µmol/L of various caspase inhibitors (zVAD, general caspase inhibitor; DEVD, caspase-3 inhibitor; LEHD, caspase-9 inhibitor; IETD, caspase-8 inhibitor) for 24 h. The number of round cells was assessed under each condition and expressed as percentage of maximum determined in the presence of SN-38 and UDCA and in the absence of the caspase inhibitors. The number of round cells determined in the presence of SN-38 alone was subtracted from that of SN-38 + UDCA. **, P < 0.001; * P < 0.01, significantly different from control.

 
Role of JNK on the Combined Apoptotic Effect of SN-38 and UDCA
JNK is one of the MAPKs implicated in camptothecin-induced apoptosis. To investigate the possible involvement of the MAPKs in SN-38- and UDCA-induced apoptosis, the phosphorylation of ERK, p38, and JNK was determined by Western blotting using specific antibodies against the respective phosphorylated and total form of these kinases. These three kinases were phosphorylated to a certain extent even in untreated HT-29 cells. Following the addition of 0.5 µmol/L SN-38, the activation of ERK and p38 was observed as early as ~5 to 15 minutes and peaked at ~1 hour, whereas the phosphorylation of JNK was relatively delayed starting at ~30 minutes and reaching a plateau at ~2 hours (data not shown). This phosphorylation of JNK lasted for at least 6 hours after addition of SN-38 (Fig. 7A ) and then gradually decreased over time to control level by 24 hours (Fig. 7B and C). In the simultaneous presence of SN-38 and 100 µmol/L UDCA, the phosphorylation level of JNK remained elevated at least up to 24 hours (Fig. 7B and C). Next, we used specific JNK (SP600125) and ERK (PD98059) inhibitors. These inhibitors almost completely inhibited JNK and ERK phosphorylation, respectively, as determined by Western blotting (Fig. 7C). Under these conditions, SP600125 prevented the potentiation of SN-38-induced apoptosis by UDCA (Fig. 7D). Taken together, the results of this study suggest that one of the key mechanisms by which UDCA increased SN-38-induced apoptosis includes enhanced and/or stabilization of JNK phosphorylation and activation.


Figure 7
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  		Figure 7. Role of JNK on the combined apoptotic effect of SN-38 and UDCA. A, HT-29 cells were incubated with 0.5 µmol/L SN-38 for 2 h. After removal of SN-38, the cells were further incubated without and with 100 µmol/L UDCA. Phosphorylated (P-JNK) and total (JNK) JNK expression was determined over time in the absence and presence of either SN-38 or SN-38 + UDCA. B, ratio between phosphorylated JNK and total JNK was calculated according to the densitometric analysis at 24 h. C, HT-29 cells were incubated in the presence of either 50 µmol/L PD98059 (P) or 25 µmol/L SP600125 (S) before the addition of 0.5 µmol/L SN-38. After removal of both the respective ERK and JNK inhibitors and SN-38, the cells were further incubated for 24 h with 100 µmol/L UDCA in the presence of the same respective inhibitor. The phosphorylation of ERK1/2 (P-ERK) and JNK was determined by Western blotting using specific anti-phosphorylated antibodies. Representative of three independent experiments. D, apoptotic cell death was determined by the morphologic identification of apoptotic cells by fluorescence microscopy using the conditions described in (C) and by using Hoechst 33258. * P < 0.01 compared with the same condition but without SP600125.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that the bile acid, UDCA, promotes the apoptotic response induced by SN-38 not only in colon adenocarcinoma-derived HT-29 and LS174T cells but also in the hepatocarcinoma HepG2 cell line. Addition of concentrations ≥50 µmol/L UDCA after, rather than before or during, SN-38 administration increased DNA damage as well as decreased {Delta}{Psi}m resulting in an increased SN-38 induced apoptosis at least in HT-29 cells. UDCA had little apoptotic effect on its own; furthermore, under these conditions, UDCA did not affect the SN-38-induced alteration of the mRNA level of key proteins involved in either apoptotic or cell cycle pathways. Inhibition of caspase-3 and caspase-9 but not caspase-8 either partially or almost completely prevented the UDCA-induced apoptotic effect. These results contrast to reports supporting an antiapoptotic effect of UDCA against a variety of stimuli (15, 24). In these studies, the protective effect of this bile acid was attributed to the stabilization of the mitochondrial structure, thereby preventing the loss of cytochrome c (15, 25). Although there is no clear hypothesis to explain these differences, the present results are consistent with findings suggesting that UDCA enhances efficacy of photodynamic therapy on cancer cells (19). In the present study and that by Kessel et al., one of the clear effects of UDCA is to enhance the mitochondrial damage of the chemotherapeutic agents. Furthermore, the activation of JNK could be one of the reasons for our observed significant effect of UDCA when present after SN-38 exposure, whereas Kessel et al. reported the requirement for UDCA to be present before photoirradiation (19).

Apoptosis and necrosis can be distinguished morphologically (26). The common apoptotic markers, such as chromatin condensation, DNA fragmentation, apoptotic bodies, and caspase activation, are absent in necrotic cells (27). Once the apoptotic signal has been induced, the cell is rapidly cleared by phagocytosis without spilling its intracellular content. However, when apoptotic cells escape clearance, which is mostly the case in cell culture, the cells may present a loss of membrane permeability associated with late-stage apoptosis (termed secondary necrosis; ref. 28). Therefore, one of the possible mechanisms to explain the increased apoptosis and reduced necrosis in the presence of SN-38 and UDCA includes the UDCA-induced alteration of the apoptotic process and secondary necrosis formation. Indeed, UDCA could be preventing this loss in membrane permeability observed in secondary necrosis, because this bile acid has been shown to protect membranes against damage by hydrophobic and more toxic bile acids (29). However, the significant further decrease in colony growth observed in the presence of SN-38 and UDCA would rule against this hypothesis.

The observations using confocal laser scanning microscopy revealed the process of SN-38-induced apoptotic cell death and its enhancement by UDCA in HT-29 cells. Our results indicated that the detachment of the cells from the culture plate is an early event in apoptosis, which is consistent with previous reports using various colonic adenoma cell lines (30). In the presence of SN-38 and UDCA, the majority of the floating cells were externalizing phosphatidylserine, whereas <3% of the attached cells were phosphatidylserine positive. Deprivation of the cells from anchoring to substrate leads to rapid cell death. This form of apoptosis has been termed anoikis. Gunthert et al. observed an early detachment of the HT-29 cells from the plate in Fas-induced apoptosis (31). In this report, the authors proposed the shedding of cell surface adhesion molecules, which occurred at an early stage of Fas-dependent apoptosis, contributed to the active disintegration of the cells. However, the induction of the disappearance of these cell surface adhesion proteins may not be a Fas-specific phenomenon and may be induced by SN-38 and potentiated by UDCA.

Shrinkage of the cells, which is also generally considered as an early morphologic change in the apoptotic process, was not observed in the present study. In fact, most of the cells treated with either SN-38 or SN-38 and UDCA showed an increased cell volume, which was independent of the cell density. This precludes the possibility that the increase in size is due to the death of the surrounding cells and the increased space availability. However, although the number of semidetached cells and apoptotic bodies was increased, this increase in cell volume with SN-38 was not modified by UDCA. These data support the likelihood that the blockade of the cell cycle in the S and G2-M phases by SN-38 was responsible for the increase in cell volume as suggested previously (9). This is also consistent with the lack of effect of UDCA on SN-38-induced cell cycle alteration reported in the present study.

Release of cytochrome c from the mitochondria into the cytosol results in the activation of the caspase adaptor Apaf-1 and procaspase-9, which form a holoenzyme complex termed "apoptosome." Caspase-9 in context with this holoenzyme activates downstream caspases, most importantly caspase-3, which results in DNA fragmentation and apoptosis (32–34). In addition, the direct oligomerization of the Fas receptor (CD95/Apo-1) leads to the initial activation of the caspase cascade starting from caspase-8 (35). Recent reports indicated that bile acids can induce hepatocyte cell death via ligand-independent oligomerization of Fas and activation of the death receptor pathways (36). In addition, certain bile acids have been shown to activate a phosphatidylinositol 3-kinase/protein kinase C{zeta}/nuclear factor-{kappa}B survival pathway (37). This latter activation can oppose the Fas-dependent apoptosis and therefore can inhibit the inherent cytotoxicity of bile acids. The present study supports the involvement of caspase-9 rather than caspase-8 in the UDCA-induced potentiation of SN-38-induced apoptosis, because (a) the SN-38-induced caspase-3/9 activity is facilitated by UDCA and (b) a specific caspase-9 but not caspase-8 inhibitor can, at least partially, reduce the UDCA effect. These findings suggest the simultaneous activation of various apoptotic pathways by SN-38, whereas the involvement of UDCA is mostly limited to the caspase-9-dependent pathway. Taken together, the present study suggests that the effect of UDCA is correlated with mitochondrial membrane depolarization, and the promotion of the apoptotic signaling damage induced by SN-38, and is most probably independent from the Fas-associated apoptotic process.

SN-38 is the most potent metabolite of CPT-11 and thought to be a major player in the antitumor action of CPT-11 (6). It is known that the inhibition of DNA topoisomerase I by SN-38 leads to apoptotic cell death in various cell lines, including the adenocarcinoma LS174T colon cancer and hepatocellular HepG2 cell lines (7). The fact that SN-38 decreases the mRNA expression level of the antiapoptotic protein Bcl-xL but not that of either Bcl-2 or Bax is of interest because it has been reported previously that 10-hydroxycamptothecin induced apoptosis in HepG2 cells by decreasing Bcl-2 and Bax (38). The results of the present study are more in line with those of Magrini et al. who have shown that CPT-11 decreased Bcl-xL protein expression level without affecting that of both Bcl-2 and Bax in colonic HCT116 p53–/– cells (10). These authors suggested that the decrease in Bcl-xL known to inhibit Bax resulted in the integration of the liberated Bax into the mitochondrial membrane and the apoptosis induction (10). It is also worthwhile to mention that SN-38 induced a decreased mRNA expression level of p16INKA and c-fos. Indeed, in a previous study, Fukuoka et al. have reported that the apoptotic effect of CPT-11 was p16 dependent and was increased in A549 cells transfected with p16 (39). There are only a few studies that have focused on CPT-11 and c-fos. The study by Singh et al. suggests that there is an improved patient survival when c-fos expression is high (40). Therefore, the level of expression of both p16 and c-fos may have an important role in cells refractory to CPT-11, and further study of the role of these proteins in the CPT-11-induced cell death should be worthwhile. The stimulatory effect of various bile acids on c-fos mRNA expression and the lack thereof of UDCA has been reported previously (41) and is supported by results of the present study. Furthermore, the complete lack of effect of UDCA on the alteration of any of these mRNA levels supports a post-transcriptional effect of this bile acid.

The proapoptotic role of prolonged JNK activation has been reported recently in various colonic cancer cell lines, including HT-29 (42–44). Indeed, studies using target gene disruption have established that the JNK signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis (43). Previous reports also revealed the activation of JNK by several chemopreventive agents, including SN-38, facilitating apoptosis in cancer cells (42, 44, 45). The intermediate signaling moiety between JNK activation and cytochrome c release may be linked to the Bax protein, as activated JNK fails to induce apoptosis in cells deficient of members of the proapoptotic Bax subfamily of the Bcl-2-related proteins. In the present study, the SN-38-induced JNK activation was further prolonged by the presence of UDCA, and pretreatment with SP600125, a specific JNK inhibitor, led to a substantial decrease in apoptotic cell death induced by SN-38 and UDCA. These findings suggest that the JNK pathway plays a pivotal role in SN-38- and UDCA-induced cell death. JNK phosphorylation by UDCA may not occur only after SN-38 stimulation but rather through a direct mechanism because the long-term incubation with UDCA alone also induces the JNK phosphorylation to a certain extent. It should also be noted that the simultaneous phosphorylation of all three major MAPK (JNK, ERK, and p38) pathways, which have different and, in some cases, opposite biological functions, suggests that the balance and integration of the MAPK pathways may modulate the commitment of the cells to either apoptosis or survival following external stimuli. Indeed, the extensive interaction among all three MAPKs has been reported in various tissues and cells and is relevant to cancer therapy (see ref. 46 for review). In the present study, JNK phosphorylation by SN-38 plus UDCA was further increased in the presence of an ERK inhibitor supporting the presence of cross-talk between these two pathways in HT-29 cells and the predominance of JNK-induced cell death when stimulated with SN-38 plus UDCA. It is possible that SN-38 can induce simultaneous JNK and ERK phosphorylation and the balance between these two pathways determines the final fate of the cell. UDCA facilitation of JNK phosphorylation may in turn tip the balance between these pathways over time and enhance apoptosis.

In man, the biliary bile acid concentration ranges from 5 to 40 mmol/L, whereas the portal and systemic bile acid concentration ranges from 30 to 100 and 1 to 3 µmol/L, respectively. The oral administration of 150 to 600 mg UDCA (0.38–1.53 mmol) results in a significant increased UDCA concentration representing >50% of the total biliary bile acid concentration (47) and reaches a peak plasma concentration of up to 16 µmol/L in healthy volunteers (48). Furthermore, although the majority of the bile acid pool is reabsorbed from the small intestine, >5% escape the enterohepatic circulation and are found in the colon and feces. Therefore, although difficult to assess with accuracy, one could expect to see UDCA concentrations of 0.1 to 1 mmol/L in the colon. However, the bile acid can be dehydroxylated by bacterial enzymes, which would further decrease the intestinal UDCA concentration. To this effect, Makino and Nakagawa have reported that although UDCA was still detectable in the feces a large portion had been 7ß-dehydroxylated to lithocholic acid (49). In addition, it is quite possible that the colonic UDCA concentration could be significantly increased if this bile acid was administered rectally. Together, these results support the possibility of achieving UDCA concentrations, from the combined apical and basolateral poles of the colonocytes following UDCA administration, as shown in the present study to be able to stimulate SN-38-induced apoptosis. Furthermore, it is worthwhile mentioning that, as previously shown clinically by Takeda et al., oral administration of UDCA, at least when combined with magnesium oxide and bicarbonate, did not increase the overall toxicity of CP-11/SN-38 (50).

In summary, UDCA increases the apoptotic and decreases the necrotic effects of SN-38 in the various adenocarcinoma cell lines, including HT-29. This effect of UDCA involves mitochondrial membrane depolarization and activation of caspase-3 and caspase-9 and these actions are mediated, at least in part, through JNK activation. Taken together, these results support a beneficial effect of UDCA by increasing the targeted apoptotic-associated cell death. By facilitating apoptosis over necrosis, UDCA could be preventing or at least decreasing the associated inflammatory response as well as facilitating wound healing. However, the clinical relevance of UDCA as an enhancer of chemotherapeutic lethality remains to be confirmed.


   Footnotes
 
Grant support: Daiichi Pharmaceutical Co. Ltd., Yakult Honsha Co. Ltd., and Mitsubishi Welpharma Co. Ltd. and NIH grant R01 DK56108 (B. Bouscarel).

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

Note: Presented in part at the Annual Meeting of the American Gastroenterological Association in May 2001 and Annual Meeting of the AACR in July 2003.

Received 4/ 6/05; revised 10/14/05; accepted 10/28/05.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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