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Tumor Biology |
Klinik und Poliklinik für Innere Medizin I, Universitätsklinikum Regensburg, 93042 Regensburg, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation of colorectal cancer cell lines with physiological concentrations of deoxycholic acid led to a dramatic induction of apoptosis. Caspase cleavage and caspase activation occurred as early as 30 min after the addition of deoxycholate. Caspase-2 (Ich-1, Nedd2), caspase-3 (CPP-32, YAMA, Apopain), caspase-7 (Mch-3, ICE-LAP-3), and caspase-8 (FLICE, Mach-1, Mch5) are activated in HT-29, whereas caspase-1 (ICE) remained intact. Caspase activation and cellular apoptosis induced by bile salts were reversed by broad spectrum and selective caspase inhibitors. As opposed to hepatocyte death mediated by bile acids, CD95 was not involved in deoxycholate-induced apoptosis. The cytoprotective effect of ursodeoxycholic acid in hepatocytes or other tumor cell lines, which is mediated by inhibiting the mitochondrial permeability transition, was not observed in colon cancer cell lines as well. This points to distinct intracellular functions of ursodeoxycholate in different cancer cell types.
Here we describe the specificity of bile salt-induced apoptosis in colon cancer cell lines. Differences from hepatocytes are shown. Bile acid-specific caspase activation is part of the apoptotic pathway induced by bile salts in colon cancer cell lines. Furthermore, a lack of cytoprotective function of ursodeoxycholate in these cells is demonstrated. Our data raise questions as to the role of bile salts in colorectal carcinogenesis.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The observation of hepatocyte mitochondrial swelling and impaired state
III respiration in bile duct-ligated rats prompted further biochemical
characterization of the molecular pathways (6)
, such as
disruption of the mitochondrial respiratory chain with reversible ATP
depletion (7)
and mitochondrial permeability transition
(8)
. Activation of an intramitochondrial calpain-like
protease reportedly coincides with the mitochondrial permeability
transition attributable to toxic bile salts in hepatocytes
(7)
. Inhibitors of cystein proteases but not serin
proteases reversed this effect (9)
. It was hypothesized
that this protease might regulate mitochondrial permeability transition
by processing proteins forming the mitochondrial permeability
transition pore or proteins regulating the function of this pore
(10)
. Direct activation of the protein kinases C 
and
by glycochenodeoxycholate positively correlates with bile
salt-induced apoptosis and inhibitor studies with protein kinase C
antagonists, such as chelerythrine, suggested that protein kinase C
might play a role in apoptotic signaling of bile salts in hepatocytes
(11)
.
In a recent report, direct oligomerization of the Fas receptor (CD95/Apo-1) has been suggested as the primary causative mechanism of bile salt-mediated hepatocyte apoptosis. This oligomerization led to the activation of the caspase cascade (caspase: cysteinyl aspartic acid protease) starting from caspase-8 (FLICE), which is the first caspase activated in Fas-mediated apoptosis (12 , 13) . Caspases are responsible for morphological and biochemical changes occurring in cells undergoing apoptotic cell death. The caspases differ in their capacity to induce the caspase cascade necessary for these changes. The ICE-like caspases (e.g., caspase-1 or interleukin-1 converting enzyme) rather fulfill proinflammatory tasks, whereas other caspases are clearly proapoptotic, e.g., caspase-3 (CPP-32, Yama, Apopain). Caspases exhibit selectivity in their function attributable to their substrate specificity. An increasing number of intracellular caspase substrates has been identified, and it is believed that the induced destruction of these substrates causes the apoptotic phenotype in cells (14) .
In the present study, we characterized the kinetics of bile salt-induced apoptosis in colon cancer cell lines. The specificity of death induction by toxic bile salts is demonstrated. Activation of the caspase cascade and its role in bile salt-mediated apoptosis is described. Differences to bile acid-induced hepatocyte death are identified.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of Apoptosis.
The respective cell lines were seeded in six-well plates. Cells grew to
subconfluency. For the induction of apoptosis, new media containing the
respective amounts of the water-soluble sodium salts of DC (Boehringer,
Mannheim, Germany), CDC, GC, GDC, C (Sigma, Deisenhofen, Germany), and
UDC (Calbiochem, Bad Soden, Germany) were added. The bile salts
were sonicated for 30 min at 50°C prior to usage. Two µg/ml of the
mouse monoclonal anti-Fas (CD95/Apo-1) antibody CH-11 (Upstate
Biotechnology, Munich, Germany) were added where necessary. The broad
spectrum tripeptide caspase inhibitor z-VAD-fmk and the caspase-1
inhibitor YVAD-CHO (100 µM; both Bachem, Heidelberg,
Germany) and UDC (500 µM) were added 60 min prior to
incubation with bile salts at the indicated concentrations.
DNA Fragmentation.
Low molecular weight DNA was extracted using a hypotonic lysis buffer
consisting of 50 mM Tris, 10 mM EDTA, and 0.5%
n-lauroylsarcosine supplemented with 20 µg/ml RNaseA for
1 h at 37°C. Four hundred µg/ml of proteinase K was added, and
the solution was incubated at 65°C for 2 h on a shaker. DNA was
precipitated in ice-cold 100% ethanol, 2.5 M
ammonium acetate and 1 mM magnesium chloride
overnight. After centrifugation for 30 min at 15,000 rpm, the DNA
pellet was resuspended in 30 µl of deionized water. Standard loading
buffer (1 µl) was added, and samples were run on a 1% agarose gel
containing 0.1% ethidium bromide in TAE buffer (Tris 40
mM, sodium acetate 20 mM,
and 1 mM EDTA, pH 8.0).
Flow Cytometry.
Apoptotic cells from the medium supernatant and nonapoptotic adherent
cells were collected and pelleted at 2,000 units/min. Pellets were
washed in PBS, and DNA propidium iodide staining was performed using
the CycleTest Plus DNA Reagent kit according to the manufacturer’s
recommendations (Becton Dickinson, San Jose, CA). The samples
were analyzed using a an EPICS XL-MCL (Coulter Immunotech, Hamburg,
Germany) flow cytometer. Quantitation of the
sub-G1 area was performed with the WinMDI Program (J. Trotter).
CD95 surface expression was detected with the anti-CD95 antibody UB2 (PharMingen, San Diego, CA). Adherent cells were collected and pelleted at 2,000 rpm. The resulting pellet was resuspended in ice-cold PBS/2% FCS and incubated at 4°C for 30 min. The cells were again pelleted, resuspended in 200 µl of PBS/2% FCS and stained with 10 µl of the mouse anti-CD95-FITC antibody at 4°C for 1 h prior to flow cytometric analysis.
Western Blotting.
Cells were lysed in a buffer containing 4 mM HEPES, 320
mM sucrose, 1 mM EDTA, 0.1 mM DTT,
1 mM phenylmethylsulfonyl fluoride, 1.5 mM
pepstatin, 2 mM leupeptin, 0.7 mM aprotinin,
and 0.1% CHAPS on ice for 20 min. After sonication for 10 s,
protein extracts were centrifuged for 20 min at 15,000 units/min. The
protein concentration in the supernatant was determined using the BCA
test (Sigma). Cytosolic extracts were loaded and run on 15% SDS-PAGE
gels under reducing conditions. After electrophoresis, protein was
transferred to a polyvinylidene difluoride membrane. Membranes were
blocked with 5% nonfat milk in PBS and probed with 1:3000 dilutions of
the monoclonal mouse antihuman antibodies anti-caspase-1 (Upstate
Biotechnology, Lake Placid, NY), anti-caspase-2, anti-caspase-8
(PharMingen, San Diego, CA), anti-caspase-3, and anti-caspase-7
(Transduction Laboratories/Dianova, Hamburg, Germany), and anti MAPK-1
(Santa Cruz Biotechnology, Santa Cruz, CA) in 5% nonfat milk/PBS.
Immunoblots were incubated with 1:5000 diluted horseradish
peroxidase-conjugated antimouse IgG (Dianova) using the Enhanced
Chemiluminescence Plus (ECL Plus) Western blotting detection system kit
(Amersham Pharmacia Biotech Europe, Freiburg, Germany) and exposed to
Hyperfilm (Amersham Pharmacia Biotech Europe, Freiburg, Germany).
Caspase Activity Assay.
Caspase activation was determined from cytosol of HT-29 cells treated
with bile acids for the indicated times. The colorimetric
activity assays were performed with a commercially available caspase
assay kit (Biomol Research Laboratory, Plymouth Meeting, PA)
according to the manufacturer’s recommendations. In brief, after the
respective treatment, cells were collected and briefly spun down. Cells
were lysed [50 mM HEPES (pH 7.4), 0.1% CHAPS, 1
mM DTT, and 0.1 mM EDTA], nuclei were removed
(10,000 rpm for 10 min), and the cytosolic preparations were quickly
frozen and stored at -80°C until usage (maximum, 2 weeks). Equal
amounts of cytosolic protein were added to the assay buffer [50
mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10
mM DTT, 1 mM EDTA, 10% gycerol, and caspase-3
substrate Ac-DEVD-pNA] in 96-well ELISA plates. The absorbance
at 405 nm was quantified with an ELISA plate reader.
Microscopy.
Cells were seeded in petriPerm Culture "hydrophil" dishes (Heraeus,
Munich, Germany) and grown to subconfluency. Apoptosis was induced as
described above, and the dishes were placed under an inverse
fluorescence microscope (Axiovert S 100, Zeiss, Germany) with an
incubation unit. Cells were filmed with a cooled CCD camera, and the
generated photographs were analyzed using Metamorph Software (Visitron
Systems, Puchheim, Germany). Morphological criteria used to identify
apoptotic cells were membrane blebbing, cellular shrinkage, and nuclear
condensation as well as formation of apoptotic bodies.
CD95 RT-PCR.
Total RNA of SW480 and SW620 cells was extracted using the RNeasy kit
(Qiagen, Hilden, Germany). Quality and quantity of RNA were analyzed
measuring absorption at 260 and 280 nm, respectively. RT-PCR was
performed using the RETROscript kit (Ambion, Austin, TX) to generate
cDNA with random decamer primers according to the manufacturer’s
recommendations. The oligonucleotides used for the PCR amplification
were as follows for CD95: sense strand, 5'-GACAAAGCCCATTTTTCTTCC-3';
and the antisense strand, 5'-ATTTATTGCCACTGTTTCAGG-3'. To test cDNAs
for representation and full-length genes, PCR with a 3' ß-actin, 2K
clathrin, and glyceraldehyde-3-phosphate dehydrogenase primer set from
the Gene Checker kit (Invitrogen, Leek, the Netherlands) was performed
according to the manufacturer’s protocol. The PCR for CD95 comprised
30 cycles with denaturing at 94°C for 60 s, annealing at 53°C
for 60 s, and extension at 72°C for 120 s. The PCR for the
genes from the Gene Checker kit comprised 25 cycles with denaturing at
94°C for 30 s, annealing at 55°C for 30 s, and extension
at 72°C for 30 s. The reactions were performed in a
TRIO-Thermoblock (Biometra, Goettingen, Germany). The PCR products were
then subjected to 2% agarose gel electrophoresis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). DNA laddering, a typical sign of apoptosis, was
detectable 1 h after addition of 500 µM DC
to HT-29 with increasing intensity over time (Fig. 1B
). Flow
cytometric analysis of apoptosis again showed a strong time-dependent
and concentration-dependent response after DC (Fig. 1C
).
Concentrations as low as 100 µM DC induced
detectable apoptosis after an incubation time of 90 min as well,
however, to a much lower degree.
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),
even at higher concentrations (up to 1 mM;
data not shown). Hence, all three apoptosis detection methods point to
apoptotic rather than necrotic cell death, and apoptosis was
specifically induced by toxic (hydrophobic) bile salts such as DC and
CDC, whereas the conjugates of DC as well as the trihydroxy bile salt C
and its conjugates do not induce apoptosis in colorectal cancer cell
lines. Addition of the protein synthesis inhibitor cycloheximide (50
µg/ml) revealed that DC-induced apoptosis is not protein
synthesis dependent (data not shown).
The recent observation that bile salt-induced apoptosis in hepatocytes
is mediated via the Fas receptor signaling pathway (12)
prompted us to compare the kinetics of bile salts and anti-Fas-mediated
apoptosis in HT-29, which is a Fas-positive cell line. The
anti-Fas-mediated induction of apoptosis in HT-29 requires pretreatment
with IFN-
because of an IFN--mediated increase in the expression
of Fas on HT-29 cells (15)
. In our observations, the
apoptotic response in SW480 cells treated with the agonistic anti-Fas
(Fas/Apo-1) antibody CH-11 (1 µg/ml) was negligible at time points
when almost all DC-treated cells were clearly apoptotic (Fig. 2A
). We screened cancer cell lines for CD95 expression.
Although the SW480 cell line is highly CD95 positive, the SW620 cell
line was CD95 negative in RT-PCR as well as by flow cytometric analysis
(Fig. 2B
). However, incubation of both cell lines with DC
had a comparable rapid proapoptotic effect (Fig. 2C
).
Together, these data indicate that DC is a potent inducer of apoptosis
in colon cancer cells, whereas the proapoptotic effect of the agonistic
anti-Fas antibody CH-11 is negligible at time points when DC caused
full-blown apoptosis. These data suggest that DC does not exert its
proapoptotic effect by recruiting the CD95 signaling cascade in colon
cancer cell lines.
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shows typical time courses of caspase cleavage in Western
blots. Incubation of HT-29 with 500 µM DC
resulted in cleavage of the proapoptotic caspases caspase-2 (Ich-1,
Nedd2), caspase-3 (CPP-32, YAMA, Apopain), caspase-7 (Mch-3,
ICE-LAP-3), and caspase-8 (FLICE, Mach-1, Mch5). Therefore, all
investigated proapoptotic group II caspases (caspase-2, caspase-3,
caspase-7) and group III caspases (caspase-8) undergo bile salt-induced
cleavage. In contrast, caspase-1 (ICE), a proinflammatory group I
caspase, was not cleaved in our experiments (Fig. 3B
). A
Western blot of MAPK-1 proved that protein amounts of noncaspase
substrates remained constant over time after treatment of HT-29 with DC
(Fig. 3B
).
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). The caspase-1 like caspase inhibitor
YVAD-CHO did not inhibit apoptosis (Fig. 4A
). Hence, the
proapoptotic members of the caspase family of cysteine proteases are
functionally involved in the apoptotic response of colorectal cancer
cells to bile salts, whereas the proinflammatory member caspase-1 (ICE)
is not.
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). Concentrations up to 1
mM of UDC lacked any contra-apoptotic effects as
well (data not shown), whereas DC-mediated apoptosis was efficiently
suppressed by UDC in Hep-G2 cells. UDC itself had no proapoptotic or
caspase-activating effect at the indicated time points (Fig. 4
,
A–C). These data suggest that UDC effects differ in hepatic
and colon tumor cell lines.
To determine whether the apoptosis-inhibiting capacity of
z-VAD-fmk correlates with caspase cleavage in DC-treated HT-29 cells,
we analyzed caspase cleavage in Western blots. z-VAD-fmk almost
completely inhibited caspase-3 and caspase-8 cleavage (Fig. 4D
), supporting the functional importance of caspase
activation in DC-mediated apoptosis.
After the detection of bile salt-induced cleavage of caspases in HT-29,
we investigated the activation of caspase-1 and caspase-3 in the
cytoplasm of bile salt-treated HT-29 cells by colorimetric assays with
substrates indicating caspase-1 and caspase-3 like activity. The
results presented in Fig. 5
point to activation of caspase-3, whereas caspase-1 is not activated.
Again, the functional role of caspase-3 and the lack of caspase-1 in
apoptotic signaling induced by DC in HT-29 cells was emphasized.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epidemiological studies, results from in vitro and in vivo mutagenesis experiments, and data on bile salt-induced proliferation and signaling (16) imply a cocarcinogenic effect for certain bile salts in the development of colorectal cancer (1 , 2) . DC constitutes the major fraction of bile salts in the colon. Because of its hydrophobicity, the largest portion is found in the feces, but considerable amounts up to 700 µM DC was measured in fecal water (17, 18, 19, 20) . This portion, at least theoretically, can get into contact with colonic epithelial cells. We investigated the effect of different bile salts on various colon epithelial cancer cell lines. Induction of apoptosis in all examined cell lines by the unconjugated deoxy derivative of C, DC was observed, suggesting that dehydroxylation is critical for apoptosis induction. Conjugation with glycine residues resulted in a complete lack of cytotoxicity, most likely attributable to decreased permeability of the cytoplasmic membrane for these bile salts in colon cancer cells. The fact that all examined cell lines dramatically responded to bile salts within 30–40 min raises questions as to the postulated resistance of ex vivo epithelia from colonic epithelial biopsies to bile salt-induced apoptosis (21) . The authors propose the selection of apoptosis-resistant colonic epithelial clones that subsequently undergo genetic alterations resulting in oncogenic transformation. The concentrations of DC used in our experiments were 100–500 µM, whereas Bernstein et al. (21) incubated colonic epithelial cells in 1000 µM DC. Hence, there is an obvious contradiction that cannot be solved by the mere fact that we used tumor cell lines as opposed to the biopsies from Bernstein et al. (21) . Our "bile acid-resistant" cancer cells should have responded to a much lower degree according to the model of Bernstein et al. (21) . At present, we investigate the DC effects in ex vivo colon crypt epithelial cells in a cell culture model recently established in our laboratory (22) to gain further insight into the biology of bile salts on these ex vivo cells.
The signal transduction of bile salt-mediated apoptosis has not yet
been investigated in colon cancer cells. The data demonstrated in this
report propose early and strong DC-dependent activation of the
caspase-2, caspase-3, caspase-7, and caspase-8. The activation of
caspase-8 was surprising, because activation of caspase-8 is usually
observed upon receptor-mediated apoptosis (CD95, TNF-R1). This caspase
has a death effector domain and binds to the death effector domain of
adaptor proteins such as FADD and FLASH (23
, 24)
. Binding
of caspase-8 to death receptor-bound adaptor molecules with subsequent
oligomerization leads to activation of caspase-8. Hence, activation of
caspase-8 in our case was not expected and is not yet clarified. A bile
salt-mediated up-regulation of the CD95 ligand could lead to autocrine
suicide in the CD95-positive HT-29 cells but is rather unlikely because
addition of a CD95-blocking antibody did not inhibit DC-mediated
apoptosis in
HT-29.5
HT-29 and, in our hands also the CD95-positive cell line SW480, do not
undergo apoptosis upon addition of the agonistic anti-Fas antibody
CH-11. We used up to 2 µg of this antibody to induce apoptosis
without any proapoptotic effect. In fact, only after preincubation with
IFN- did HT-29 up-regulate CD95 and thereby become sensitized to
CH-11-dependent apoptosis (15
, 25) . Furthermore, our
CD95-negative cell line SW620 undergoes bile salt-induced apoptosis to
an equal degree as the CD9- positive cell line SW480, rather excluding
CD95 as an effector of bile salt-induced apoptosis. Interestingly,
recent work demonstrated activation of caspase-8 independently from
death receptors (26, 27, 28)
. One might speculate that either
caspase-8 is directly activated by bile salts or that caspases that are
usually described as "upstream" of the perturbation of the
mitochondrial transmembrane potential can be activated by caspases that
are "downstream" of the mitochondrion, e.g., the
initiator caspase, caspase-9. Caspase-9 activation occurs upon
mitochondrial permeability transition when it becomes part of the
apoptosome, a multimolecular complex of cytochrome c,
apaf-1, dATP, and caspase-9 itself. These data suggest additional
mechanisms by which bile salts cause cell death in colon tumor cell
lines as compared with hepatocytes.
In this respect, it is important that bile salts can directly mediate mitochondrial permeability transition in isolated mitochondria from hepatocytes (8 , 29) . The zymogen form of caspase-2 has been shown to be released from mitochondria after opening of the permeability transition pore and to be subsequently activated during the apoptotic process (30) . We were able to detect cleavage of caspase-2 in HT-29 cells treated with DC as well, indicating DC-dependent mitochondrial permeability transition in HT-29.
However, the observation that coadministration of UDC or its taurine and glycine conjugates with toxic bile salts resulted in abrogation of the cytotoxicity in hepatocytes and even in a sarcoma cell line via stabilization of the mitochondrial permeability transition pore (8 , 29) was not reproducible in our colon cancer cell lines. Therefore, UDC differs in its capacity to protect hepatic cancer cells and colorectal cancer cells from apoptosis. Bile salt-mediated induction of the mitochondrial permeability transition in colorectal cancer cell lines and the lack of UDC-mediated inhibition of the mitochondrial permeability transition are under current investigation.
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FOOTNOTES |
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1 This work was supported by Grant DFG-RO 1236/4-1
from the German Research Foundation. 
2 The first two authors contributed equally to the
publication and therefore share first authorship.
3 To whom requests for reprints should be
addressed, at Department of Internal Medicine I, University Hospital,
University of Regensburg, 93042 Regensburg, Germany. Phone:
(0) 941-944-7001; Fax: (0) 941-944-7002; E-mail: klaus.schlottmann{at}klinik.uni-regensburg.de
4 The abbreviations used are: DC, deoxycholate; C,
cholate; GDC, glycodeoxycholate; GC, glycocholate; UDC,
ursodeoxycholate; CHAPS,
3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate; MAPK,
mitogen-activated protein kinase; RT-PCR, reverse transcription-PCR;
z-VAD-fmk, z-Val-Ala-DL-Asp; Ac-DEVD-pNA,
Ac-Asp-Glu-Val-Asp-paranitroanilin; ICE, interleukin-1ß
converting enzyme; FLICE, FADD-like-ICE.
Received 10/27/99. Accepted 5/25/00.
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REFERENCES |
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