Antagonism of CD95 signaling blocks butyrate induction of
apoptosis in young adult mouse colonic cells
Yang-Yi
Fan1,
Jianhu
Zhang1,
Rola
Barhoumi2,3,
Robert C.
Burghardt2,3,
Nancy D.
Turner1,
Joanne R.
Lupton1,2, and
Robert S.
Chapkin1,2
1 Molecular and Cell Biology
Group, Faculty of Nutrition,
2 Department of Veterinary
Anatomy and Public Health, and
3 Image Analysis Laboratory,
Texas A&M University, College Station, Texas 77843
 |
ABSTRACT |
There is great
interest in utilizing butyrate as a chemopreventive agent for colon
tumorigenesis because of its ability to promote apoptosis in colon
tumor cell lines. Because CD95 (APO-1/Fas) transduces signals resulting
in apoptosis, we tested the hypothesis that butyrate-dependent
colonocyte apoptosis is mediated by this death receptor. Butyrate (1 mM) exposure for 24 h upregulated expression of Fas and
its ligand in young adult mouse colon (YAMC) cells. To delineate the
proapoptotic effect of butyrate and to avoid the confounding effects of
detachment from the extracellular matrix, adherent cell apoptosis was
monitored as loss of plasma membrane asymmetry and dissipation of
mitochondrial membrane potential (
mt) by laser
cytometry. Soluble Fas receptor protein (Fas:Fc chimera) and caspase
inhibitors (z-VAD-fmk and z-IETD-fmk) blocked butyrate induction of
apoptosis. Treatment with Fas agonistic antibody (clone Jo-2)
significantly induced cell death, indicating that Fas in colonocytes is
functional. In addition, butyrate promoted apoptosis by inducing loss
of 
mt and phospholipid
asymmetry of the plasma membrane after 12 and 24 h of exposure,
respectively, before cell detachment. Therefore, Fas receptor-dependent
signal transduction is involved in butyrate induction of apoptosis in colonocytes.
anoikis; plasma membrane asymmetry; mitochondrial membrane
potential; colon cancer; Fas; APO-1
 |
INTRODUCTION |
MAINTENANCE OF THE COLONIC epithelium requires a
dynamic equilibrium between cell proliferation and cell death (32).
Until recently, colon cancer development was thought to occur primarily through increased cell proliferation at several stages of the tumorigenic process, and little emphasis was placed on programmed cell
death (apoptosis). The emphasis has now shifted, and there is
considerable interest in the relationship between colonic apoptosis and
malignant transformation (13, 18). Inhibition of apoptosis is now
thought to be an integral component of the genesis of colorectal adenomas and carcinomas (4, 16, 31). Recent studies of humans
demonstrate that reduced apoptotic ability may predispose individuals
to increased risk for colon cancer (16).
In certain biological systems, apoptosis plays a central role in animal
development by sculpting anatomical structures and deleting unneeded
tissue (21). In these cases, apoptosis is a dominant mechanism,
involving a high percentage of cells. In contrast, in the large
intestine, apoptosis occurs with very low frequency, i.e., less than
one apoptotic cell per crypt (8, 18). Although apoptosis is not a
dominant feature in histological material, there is compelling evidence
that it is a central feature in the regulation of cell number and the
elimination of nonfunctional, harmful, or abnormal cells in the colon
(8, 18, 32, 37).
Apoptosis-signaling death receptors are characterized by an
intracellular region referred to as the death effector domain (DED),
which is required for the transmission of the cytotoxic signal (1, 34).
Currently, five distinct death receptors are known, including CD95
(APO-1/Fas), tumor necrosis factor (TNF) receptor (TNFR) 1, TNFR-related apoptosis-mediated protein, and TNF-related
apotosis-inducing ligand receptors 1 and 2 (1, 34). With regard to the
colonic mucosa, the Fas receptor is considered an important candidate
for regulating apoptosis (28, 29, 38, 42). Whereas Fas is strongly
expressed throughout the normal colonic crypt (28), it is downregulated
and progressively reduced during malignant transformation (6, 28). In
addition, colon carcinoma cells can acquire mechanisms to escape
Fas-mediated apoptosis. These include antagonism of Fas, inhibition of
Fas capping, and activation of antiapoptotic programs (42). With regard
to Fas activation, binding either to its natural ligand, CD95L (Fas-L),
or with agonistic antibodies induces apoptosis in some cells. However,
Fas-L expression is virtually absent in the normal colon (29). Thus the
biological role for Fas-mediated apoptosis in the colon is still obscure.
Butyrate, a short-chain fatty acid derived in the colon from microbial
fermentation of diet-derived complex carbohydrates, promotes
differentiation and apoptosis in a variety of colon tumor cell lines
(2, 17, 44). As a result, there is great interest in utilizing butyrate
as a chemotherapeutic and chemopreventive agent (7, 9). However, the
specific mechanisms by which butyrate induces apoptosis have not been
fully elucidated. In addition, an early feature of colonic cells
undergoing apoptosis is detachment from the extracellular matrix (18,
44). This subset of apoptosis is known as anoikis (18). The phenomenon is similar to what occurs in the colonic epithelium in vivo, and there
is good evidence that it occurs in both immortalized and colonic
carcinoma cell lines in vitro (2, 20, 44). However, it is
unknown whether butyrate induction of apoptosis is mediated by
disrupting interactions between colonocytes and the extracellular matrix.
In the present study, we utilized the young adult mouse colon (YAMC)
cell line, which was isolated from transgenic mice bearing a
temperature-sensitive mutation of the simian virus 40 (SV40) large T
antigen gene (43), to determine whether butyrate stimulation of colonic
apoptosis is mediated by CD95 (APO-1/Fas). Conditionally immortalized
YAMC cells are a relevant model to examine the molecular mechanisms by
which diet-derived factors affect relative cancer risk (2, 43). Our
results demonstrate that butyrate induces an apoptotic phenotype in
adherent colonocytes as defined by loss of plasma membrane phospholipid
asymmetry in the absence of cell leakage, dissipation of mitochondrial
membrane potential, and upregulation of Fas and Fas-L expression. In
addition, we demonstrate that there is antagonism of Fas signaling by
caspase inhibitors (z-VAD-fmk and z-IETD-fmk) and that soluble Fas
protein blocks butyrate induction of apoptosis in both adherent and
nonadherent (floating) populations of cells. Therefore Fas functions as
a transducer of butyrate-induced cell death in colonic cells.
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MATERIALS AND METHODS |
Materials.
RPMI 1640 and Hanks' balanced salt solution (HBSS) were from Mediatech
(Herndon, VA). Fetal bovine serum was from Hyclone (Logan, UT).
Insulin, transferrin, selenium, and linoleic acid were obtained from
Collaborative Biomedical Products (Bedford, MA). Glutamax and
recombinant mouse interferon-
(IFN-
) were from GIBCO BRL (Grand
Island, NY). Cellular DNA fragmentation ELISA and the cell death
detection ELISA were from Boehringer Mannheim (Indianapolis, IN).
Hamster anti-mouse Fas monoclonal antibody (clone Jo-2), annexin
V-FITC, 10× binding buffer, and propidium iodide (PI) were
obtained from Pharmingen (San Diego, CA). Hamster IgG was from Jackson
ImmunoResearch Labs (West Grove, PA). The general caspase inhibitor
(z-VAD-fmk) and caspase 8 inhibitor (z-IETD-fmk) were from Enzyme
Systems Products (Livermore, CA). Prepoured polyacrylamide gradient
gels were from Novex (San Diego, CA). Electroblotting polyvinylidene
difluoride membranes were obtained from Millipore (Burlington, MA).
Bicinchoninic acid (BCA) protein assay and SuperSignal chemiluminescent
detection reagents were from Pierce (Rockford, IL). Antibodies were
obtained from the following suppliers: rabbit anti-Fas M-20 and rabbit
anti-Fas-L C-178, Santa Cruz Biotechnology (Santa Cruz, CA); mouse
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MAB374, Chemicon
(Temecula, CA); mouse anti-SV40 DP01, Oncogene (Cambridge, MA); and
peroxidase-conjugated secondary antibodies, Kirkegaard & Perry
(Gaithersburg, MD). Rhodamine 123 was obtained from Molecular Probes
(Eugene, OR). Soluble recombinant Fas:Fc and Fas-related TNFR60:Fc were
gifts from Dr. Carl Ware, La Jolla Institute for Allergy and
Immunology. All other reagents were obtained from Sigma (St. Louis, MO).
Cell culture.
Conditionally immortalized YAMC cells were obtained from R. H. Whitehead, Ludwig Cancer Institute (Melbourne, Australia) (43). YAMC
cells (passage 10-16) were
cultured in RPMI 1640 supplemented with 5% fetal bovine serum, 1%
insulin-transferrin-selenium-linoleic acid, and 1% 200 mM
Glutamax. The medium was supplemented with 5,000 units/l
of recombinant IFN because the temperature-sensitive mutant SV40 large
T antigen gene (tsA58) is under an IFN-inducible promoter, mouse
H-2Kb class I gene (43). Cells
were cultured under permissive (33°C) or nonpermissive (39°C)
conditions as previously described (2).
Apoptosis in nonadherent cells.
Either cellular DNA fragmentation ELISAs or cell death detection ELISAs
were used to measure apoptosis in floating-cell populations (2).
Briefly, for the cellular DNA fragmentation ELISA, 30,000 cells were
seeded into 35-mm dishes and incubated for 24 h. Nonadherent cells were
rinsed off with HBSS, and adherent cells were provided fresh medium
containing 10 µM 5-bromo-2'-deoxyuridine and
incubated overnight. The following day, fresh medium containing sodium
butyrate (0, 1, or 5 mM), hamster anti-mouse Fas monoclonal antibody
(clone Jo-2; at 5 or 10 µg/ml), or hamster IgG (10 µg/ml) was added
to culture dishes, and the cells were incubated for 24 h at 33 or 39°C. The level of butyrate was selected on the basis of luminal composition data from the rodent colon, where normal butyrate levels
are in the 1-4 mM range (45). This is also the range within which
the large majority of in vitro butyrate studies have been conducted
(17, 19, 20). Above these levels, cytotoxic effects have been observed
(2, 35).
In selected experiments, cells were preincubated with the general
caspase inhibitor z-VAD-fmk (25, 50, and 75 µM) or caspase 8 inhibitor z-IETD-fmk (5 and 10 µM) 1 h before butyrate treatment. In
addition, in separate experiments, soluble recombinant human Fas
(Fas:Fc) at 2.8 µg/ml or Fas-related TNFR (TNFR:Fc) at 2.8 µg/ml
(5, 10) was coincubated with butyrate for 24 h. At the end of the
incubation period, adherent-cell viability was assessed by simultaneous
staining with fluorescein diacetate and PI (22), and floating cells
were harvested to measure levels of DNA fragmentation. Floating cells
were lysed and centrifuged at 2,000 g
to sediment intact nuclei. Supernatants containing low-molecular weight
DNA were subsequently analyzed by ELISA. Values of absorbance at 450 nm
(A450) were
normalized by the number of adherent cells per dish. For the cell death
detection ELISA, cells were seeded as described above. The following
day, instead of being prelabeled overnight with
5-bromo-2'-deoxyuridine, cells were provided fresh medium with or
without treatment and incubated for an additional 24 h.
Floating cells were harvested to measure apoptosis. Cells were washed
with medium, lysed, and centrifuged at 2,000 g to sediment cell nuclei.
Supernatants containing mono- and oligonucleosomes were subsequently
analyzed by ELISA. Values (A405) were
normalized by the number of adherent cells per dish. We have previously
demonstrated the validity of expressing apoptosis data relative to
adherent cell number (2).
Apoptosis in adherent cells.
The percentage of adherent cells undergoing apoptosis was measured by
an annexin V-FITC and PI dual-labeling technique adapted for use in our
laboratory. Cells were seeded onto four-well Lab-Tek chambered cover
glass (Nunc, Naperville, IL) at 25,000/well for 24 h; this was followed
by butyrate with or without apoptosis inhibitors (described above) for
another 24 h. Adherent cells were rinsed once with sterile PBS, and
then 1× binding buffer containing 2.5 mM
CaCl2 (diluted from 10×
binding buffer) was added to the well. Cells were subsequently
incubated with 25 µl of annexin V-FITC and 50 µl of PI for 15 min
in the dark at room temperature. After 15 min, the solution was
aspirated and the cells were gently rinsed with 1× binding buffer
twice. Binding buffer (200 µl) was added to each well, and the
dual-labeled cells were visualized by phase-contrast and fluorescence
microscopy with a Nikon Diaphot inverted microscope. For each well,
cells stained with either annexin V-FITC, PI, or both were counted from three representative fields, then normalized by the total cell number
in that same field under phase-contrast conditions. Typically, the
total cell numbers in representative fields varied between 100 and 300 cells.
Immunoblot analysis.
For time course Western blot analysis, 1 × 106 YAMC cells were cultured in
T-75 flasks in the presence of 1 mM sodium butyrate for 0, 8, or 24 h.
After culture, the medium was removed and adherent cells were washed
with HBSS three times. Cells were scraped into 3 ml of homogenization
buffer (50 mM Tris · HCl, pH 7.2, 150 mM NaCl, 1.0%
Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 2 mM EDTA, 1 mM EGTA,
50 µM NaF, 100 µM sodium orthovanadate, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 25 µg/ml pepstatin) (30, 40). Cell lysates were
passed through a 25-gauge needle three times and incubated on ice for
30 min. Cellular debris was removed by centrifugation (13,600 g, 15 min) at 4°C. The cell
extracts were aliquoted and frozen at
80°C. The protein
concentration of each extract was determined against a standard by the
BCA protein assay. Cell extracts were treated with pyronin sample
buffer and subjected to Tris-glycine gel electrophoresis by using
10-20% gradient polyacrylamide gels as described by Laemmli (24).
After electrophoresis, gels were electroblotted onto a Millipore
Immobilon-P transfer membrane with a Hoefer Mighty Small Transphor unit
(Pharmacia, Piscataway, NJ) at 400 mA for 75 min. After transfer, the
membrane was processed by the method of Davidson et al. (11). Membranes
were incubated with primary antibody (rabbit anti-Fas, rabbit
anti-Fas-L, mouse anti-GAPDH, or mouse anti-SV40) diluted in blocking
buffer at 4°C overnight. Dilution of the primary antibody was
titrated for each protein. Peroxidase-conjugated secondary antibody
(1:10,000) incubation was for 1.5 h at room temperature and was
followed by luminescence development with the SuperSignal reagent
mixture. Blots were exposed to Kodak Biomax MR film, scanned, and
quantitated with IQ software (BioImage, Ann Arbor, MI) for steady-state
level quantitation of Fas, Fas-L, GAPDH, and SV40. A range of protein concentrations for each target protein was loaded onto the gel to
ensure that the response was quantitative.
Assessment of mitochondrial membrane potential in adherent cells.
The potentiometric probe rhodamine 123 was used to provide a relative
measure of mitochondrial membrane potential
(
mt) (12) as previously
described (3). YAMC cells in two-well cover glass chamber slides were
incubated with butyrate for 0, 6, 12, or 24 h and subsequently loaded
with 2.5 µM rhodamine 123 for 15 min in an incubator at 33°C.
Cells were washed three times with culture medium without serum, the
analysis of rhodamine 123 fluorescence intensity was then
performed at an excitation wavelength of 488 nm, and the
emitted fluorescence of adherent cells was monitored at 530 nm with an
Ultima confocal microscope (Meridian Instruments, Okemos, MI). Data
from at least eight areas per well and from four wells per treatment
group were collected in each experiment.
Data analysis.
Statistical significance was calculated by one-way ANOVA. When
P values were <0.05 for the
treatment effects, means were separated by Duncan's multiple range test.
 |
RESULTS |
Butyrate induces loss of plasma membrane asymmetry in adherent
colonocytes.
To characterize YAMC cell apoptosis after butyrate treatment, we
examined phosphatidylserine (PS) externalization in adherent colonocytes. Elevated levels of apoptotic cells were detected 24 h
after exposure at the permissive temperature, 33°C (Fig. 1, A and
B). Because PS
exposure occurs early after the onset of apoptosis (1, 34), these data
demonstrate for the first time that a significant number of apoptotic
cells are seen in the adherent cell populations (Fig.
1A). Because detachment from the
matrix occurs relatively early in the process of apoptosis (15, 44),
the level of apoptosis in nonadherent (floating) cells was also
quantitated by DNA fragmentation ELISA. Butyrate treatment (24 h)
increased apoptosis in the nonadherent population as well (Fig.
1B). These data are consistent with
previous reports that colonocytes undergo apoptosis after butyrate
exposure (2, 20, 44). As a positive control, in cells grown at the
nonpermissive temperature (39°C), the numbers of adherent early-
and late-stage apoptotic cells [cells positive for annexin V and
negative for PI (annexin
V+/PI
)
and annexin
V+/PI+,
respectively] and necrotic (annexin
V
/PI+)
cells were significantly (P < 0.05)
increased (Figs. 1A and 2). Late-stage apoptosis or secondary
necrosis (annexin
V+/PI+)
is likely the consequence of an apoptotic process in culture where no
phagocytes are present to remove dying cells (41, 44). The effect of
butyrate treatment on the total percentage of apoptotic cells per
culture (adherent + floating populations) is described in Table
1. On average, there were 10-fold more
apoptotic cells in the floating-cell population than in the
adherent-cell population. However, with butyrate treatment there were
>20-fold more apoptotic cells in the floating-cell population than in
the adherent-cell population.


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Fig. 1.
Induction of apoptosis by butyrate in young adult mouse colon (YAMC)
cells. Cells were treated with butyrate (1 mM) and were incubated at
33°C (butyrate) or with vehicle and were incubated at 33°C
(control) or 39°C (nonpermissive temperature; 39°C) for 24 h.
A: adherent cells were analyzed by
annexin V-FITC and propidium iodide (PI) dual labeling as described in
MATERIALS AND METHODS.
Fluorescein-PI-labeled cells were averaged from 3 representative fields
per well, with 3-4 wells counted per experiment, and normalized by
total adherent cell number in the same field. Total numbers of adherent
cells were 81,000 ± 6,472 (control), 65,136 ± 3,041 (butyrate
treatment), and 68,560 ± 1,939 (39°C treatment). Apoptotic
index = (no. of apoptotic cells/total no. of adherent cells per
culture) × 100. Data are mean percentages of control ± SE
from 3-5 separate experiments (n = 20 wells). Different letters over bars indicate significant
differences (P < 0.05) within a
specific apoptotic phenotype. B:
Nonadherent (floating) cells were harvested, and apoptotic index was
quantitated by cellular DNA fragmentation ELISA. Apoptotic index = [mean absorbance at 450 nm
(A450)/total
no. of adherent cells per dish] × 10-5, and values are percentages
of control. Numbers of floating cells were also determined: control,
3,831 ± 310; butyrate treatment, 18,695 ± 710; and 39°C
incubation, 15,271 ± 370. Data are means ± SE from 2 separate
experiments; n = 4. Different letters
over bars indicate significant differences
(P < 0.05).
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Fig. 2.
Classification of fluorescent staining patterns in adherent cells after
24 h of butyrate treatment (magnification, ×200).
A: early-stage apoptotic (annexin
V+/PI )
cells (green). B: late-stage apoptotic
(annexin
V+/PI+)
cells (orange). C: necrotic (annexin
V /PI+)
cells (red).
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Because significant populations of adherent cells express the initial
features of apoptosis before detachment (Fig.
1A), a kinetic analysis of
butyrate-induced apoptosis was made over a 0- to 36-h incubation period
(Fig. 3). The data indicate that early
apoptotic cells (annexin
V+/PI
)
are not detected in the adherent population at 12 h.

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Fig. 3.
Kinetic analysis of butyrate-induced apoptosis. YAMC cells were treated
with 1 mM butyrate and incubated for up to 36 h. Adherent-cell
apoptosis was determined at 0, 4, 8, 12, 24, and 36 h after butyrate
treatment by annexin V-FITC and PI dual labeling (see Fig. 1 legend).
Values are normalized relative to total no. of adherent cells per
culture field (n = 4 wells).
* Significantly different (P < 0.05) from control (no butyrate).
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Butyrate exposure increases Fas and Fas-L protein levels.
We determined whether butyrate upregulates functionally important death
signaling molecules in YAMC cells. As shown in Fig. 4, A and
B, after 8 h of butyrate treatment,
Fas and Fas-L protein levels were increased by 52 and 47% of the
control, respectively. At 24 h, butyrate increased Fas and Fas-L
protein levels by 55 and 96% of the control, respectively. In
contrast, butyrate did not affect the expression of SV40 large T
antigen or the housekeeping gene, the GAPDH gene, at either time point.


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Fig. 4.
Butyrate increases Fas and Fas-L protein levels. YAMC cells were
treated with butyrate (1 mM) and incubated at 33°C for 0, 8, or 24 h. Adherent-cell lysates were harvested, and protein expression was
analyzed by immunoblotting. A:
representative immunoblot analysis of Fas, Fas-L, simian virus 40 (SV40), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
B: densitometric analysis of butyrate
effects on Fas, Fas-L, SV40, and GAPDH expression. Data were pooled
from 3 separate experiments and are expressed as means ± SE. Protein levels were standardized against the 0-h control;
n = 7-9 for each assay. Different
letters over bars indicate significant differences
(P < 0.05).
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Mitochondrial function-dependent apoptosis.
Because alterations in mitochondrial membrane potential have been
implicated in apoptosis (20), we determined whether butyrate exposure
initiates a process whereby the

mt is dissipated (Fig. 5). At both 1 and 5 mM butyrate,

mt in adherent cells was
dissipated but not collapsed after 12 h of exposure. Therefore butyrate
is capable of inducing mitochondrial damage after an initial lag period, before exposure of PS on the outer cell membrane leaflet.

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Fig. 5.
Butyrate induces dissipation of the mitochondrial membrane potential
( mt) in adherent YAMC
cells. The  mt for adherent
cells was determined at 6, 12, and 24 h after butyrate treatment (0, 1, and 5 mM) with the potentiometric probe rhodamine 123 as described in
MATERIALS AND METHODS. All values are
means ± SE and were obtained from a minimum of 8 regions per well
collected from 4 different chamber slide wells. Different letters over
bars indicate significant differences
(P < 0.05).
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Fas mediates apoptosis in YAMC cells.
Because Fas is a receptor that transduces signals resulting in cell
death, we asked how YAMC cells would respond when cultured with an
agonistic Fas antibody (Jo-2). The agonistic anti-Fas (5 or 10 µg/ml)
or control IgG was added to colonocytes. Cultures were maintained for
24 h at the permissive temperature (33°C), after which nonadherent
cells were harvested and apoptosis was measured by DNA fragmentation
ELISA (Fig. 6). Overall, Fas-mediated killing was elevated by 150-280%, in a dose-dependent manner, relative to the control. These data indicate that an apoptosis-inducing receptor, Fas, is functional in YAMC cells.

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Fig. 6.
Induction of apoptosis after treatment with agonistic Fas antibody.
YAMC cells were treated with Fas antibody (clone Jo-2) at 5 or 10 µg/ml, hamster IgG (10 µg/ml; negative control), or vehicle
(control) for 24 h. Nonadherent cells were harvested, and apoptosis was
measured by cellular DNA fragmentation ELISA. Apoptotic index = (mean
A450/total no. of
adherent cells per dish) × 10 5. Data are means from 2 separate experiments ± SE; n = 6 wells. Different letters over bars indicate significant differences
(P < 0.05).
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Butyrate-induced death of YAMC cells is blocked by soluble Fas
protein and caspase inhibitors.
The ability of butyrate and Fas antibody to induce apoptosis in YAMC
cells raised the possibility that butyrate induction of cell death is
mediated by Fas. To determine if Fas is involved in butyrate-induced
apoptosis, the neutralizing effect of a soluble form of Fas that binds
to the Fas-L on the cell surface was utilized. Cells were cultured in
the presence of a chimeric molecule consisting of the extracellular
region of human Fas and the Fc portion of human IgG (Fas:Fc), as
previously described (5). Butyrate induced 9.7- and 3.6-fold increases
in early- (annexin
V+/PI
)
and late-stage (annexin
V+/PI+)
adherent apoptotic cells, respectively, which were inhibited by soluble
Fas:Fc (Fig.
7A). As
a control, a chimeric molecule consisting of the extracellular portion
of the Fas-related TNFR and the Fc portion of human IgG (TNFR:Fc) was
coincubated with butyrate (10). Unlike Fas:Fc, this molecule had no
effect on butyrate-induced cell death. In addition, Fas:Fc antagonized
butyrate-induced apoptosis in nonadherent (floating) cells (Fig.
7B). Another means of antagonizing
Fas-mediated induction of apoptosis is to block the upregulation of key
catalytic effectors (caspases) that are activated after Fas ligation
(36). We initially determined the effect of z-VAD-fmk, a broad spectrum
polypeptide inhibitor of the interleukin-1-converting enzyme (ICE)
family of cysteine proteases (caspases) (23). Consistent with the role
of caspases in apoptosis, z-VAD-fmk pretreatment blocked the induction
of early-stage apoptosis (annexin
V+/PI
)
in adherent cells after 24 h of treatment with 1 mM butyrate (Fig.
8A). In
contrast, z-VAD-fmk only partially blocked the induction of early-stage
apoptosis (annexin
V+/PI
)
after incubation with 5 mM butyrate. This may indicate that butyrate is
capable of inducing apoptosis by more than one mechanism. Because FLICE
(caspase 8) directly interacts with the DED of Fas and is the first in
the cascade of proteases activated by Fas ligation (23, 26), we
examined the effect of the peptide inhibitor z-IETD-fmk on
butyrate-induced apoptosis. z-IETD-fmk, which inhibits caspase 8 (26),
blocked the proapoptotic effect of butyrate in both adherent-cell (Fig.
8B) and nonadherent (floating)-cell populations (Fig. 8C). In addition,
z-IETD-fmk blocked butyrate-dependent dissipation of

mt (control, 1,225 ± 39 arbitrary units; 1 mM butyrate, 962 ± 44 arbitrary units; 1 mM
butyrate-5 µM z-IETD-fmk, 1,253 ± 44 arbitrary units; for the
control vs. 1 mM butyrate values, P < 0.05; n = 16) in adherent cells
after 24 h of treatment. Therefore butyrate can induce apoptosis in
YAMC cells via a Fas-dependent mechanism.


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Fig. 7.
Butyrate-induced activation of apoptosis is blocked by soluble Fas
protein. A: cells were treated for 24 h with vehicle (control), 2.8 µg/ml Fas:Fc (Fas), 2.8 µg/ml
TNFR60:Fc (TNFR), 1 mM butyrate, a combination of 1 mM butyrate and 2.8 µg/ml Fas:Fc (But + Fas), or a combination of 1 mM butyrate and 2.8 µg/ml TNFR60:Fc (But + TNFR). Apoptosis in adherent cells was
measured by annexin V-FITC and PI dual labeling (see Fig. 1 legend).
Values are normalized relative to total adherent cells per field. Total
numbers of adherent cells in each well were 82,300 ± 5,994 (control), 72,680 ± 6,333 (Fas treatment), 82,256 ± 8,556 (TNFR
treatment), 46,200 ± 6,205 (butyrate treatment), 40,000 ± 5,393 (butyrate and Fas:Fc treatment), and 31,552 ± 5,123 (butyrate and
TNFR60:Fc treatment). Data are means from 2 separate experiments ± SE; n = 6-9 wells. Different
letters over bars indicate significant differences
(P < 0.05) within a specific
apoptotic phenotype. B: nonadherent
(floating) cells were harvested, and apoptotic index was quantitated by
cellular DNA fragmentation ELISA. Apoptotic index = (mean
A450/total no. of
adherent cells per dish) × 10 5. Data are means from 2 separate experiments ± SE; n = 5-9. Different letters over bars indicate significant differences
(P < 0.05).
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Fig. 8.
Neutralization of butyrate-induced apoptosis after treatment with
antagonistic caspase inhibitors. Values are means ± SE. Apoptotic
index = (mean
A450/total no. of
adherent cells per dish) × 10 5.
A: YAMC cells were pretreated with
vehicle (control) or 25 µM z-VAD-fmk, a general caspase inhibitor
(VAD), for 1 h and then were incubated with vehicle, 1 mM butyrate
(But), or 5 mM butyrate for 24 h. Different letters over bars indicate
significant differences (P < 0.05;
n = 6 wells from 2 separate
experiments) within a specific apoptotic phenotype.
B: YAMC cells were pretreated with
vehicle (control) or with 5 or 10 µM z-IETD-fmk, a caspase 8 inhibitor, for 1 h and then were incubated with vehicle or 1 mM
butyrate for 24 h. Apoptosis in adherent cells was measured by annexin
V-FITC and PI dual labeling (see Fig. 1 legend). Different letters over
bars indicate significant differences
(P < 0.05;
n = 6 wells from 2 separate
experiments) within a specific apoptotic phenotype.
C: YAMC cells were pretreated with 25 µM z-VAD-fmk or 10 µM z-IETD-fmk for 1 h and then were incubated
with vehicle or 1 mM butyrate for 24 h. Nonadherent cells were
harvested for apoptosis analysis by cellular DNA fragmentation ELISA.
Different letters over bars indicate significant differences
(P < 0.05;
n = 6 wells from 3 separate
experiments).
|
|
 |
DISCUSSION |
It has now been clearly established that the transformation of the
colonic epithelium to carcinomas is associated with a progressive inhibition of apoptosis (4, 16, 31). Recently, investigators have
focused on the mechanisms responsible for the reduction of apoptosis in
colon cancer (2, 4, 17, 20, 29). Interestingly, for the rat
azoxymethane colon tumorigenesis model, we have demonstrated that
apoptosis has greater prognostic value than cell proliferation in terms
of predicting which animals will develop colonic tumors (8). These data
suggest that the assessment of apoptosis deserves a place in the
armamentarium of intermediate biomarkers for colon tumorigenesis. In
this context, colon cancer is a disease strongly influenced by
environmental factors, with diet being one of the most important
modifying agents. Among dietary factors, the protective effect of
high-fiber (i.e., complex plant carbohydrate) diets has been
attributed, in part, to the production of butyrate by anaerobic
fermentation in the colon (9, 17), although this remains a
controversial area of research (25). One hypothesis to explain the
tumor suppressor activity of butyrate is that this short-chain fatty
acid induces apoptosis in a variety of colon carcinogenesis models,
providing a protective effect (2, 7, 17, 20, 44). However, the precise
mechanism of action of this putative chemotherapeutic agent has not
been fully elucidated (2, 7, 9, 17, 20).
The present study was undertaken to determine whether butyrate
stimulation of apoptosis is mediated by CD95 (APO-1/Fas). Fas is a
48-kDa transmembrane molecule whose gene belongs to the TNF/nerve growth factor receptor gene superfamily (1, 34). The
binding of Fas-L to Fas induces trimerization of the Fas receptor,
which subsequently recruits caspase 8 through a Fas-associated death domain (MORT 1) adapter (23, 26). Although Fas is strongly expressed
throughout the colonic crypt, the limited expression of Fas-L raises
questions regarding the biological role of this cell death machinery in
the colon (28, 29). Our data demonstrate that butyrate exposure
selectively induces expression of Fas-L and Fas. The Fas
receptor-ligand pair is functional, because treatment with an agonistic
Fas antibody induces apoptosis in YAMC cells.
The direct involvement of Fas and Fas-L in
butyrate-induced colonocyte apoptosis is established by the fact that
neutralizing soluble Fas protein, when provided immediately before
butyrate exposure, blocks butyrate-induced apoptosis in both adherent- and nonadherent (floating)-cell populations. In addition, apoptosis of
both adherent and nonadherent cells is inhibited by both a broad-spectrum, ICE-like protease inhibitor (z-VAD-fmk) and a caspase 8 inhibitor (z-IETD-fmk). Caspase 8 directly interacts with the activated
trimerized form of the Fas receptor (23, 26). Although the involvement
of peptide inhibitors in other pathways cannot be completely ruled out,
the antagonism of butyrate-induced apoptosis by z-IETD-fmk and the
neutralizing soluble Fas protein Fas:Fc is consistent with the direct
involvement of the Fas receptor.
It has been speculated that butyrate initiates an apoptotic phenotype
in colonocytes by disrupting interactions between cells and the
extracellular matrix (18, 44). As expected, with butyrate exposure,
YAMC cells lose their adherence to the substratum and floating cells
undergo DNA fragmentation (Table 1). To delineate the direct
proapoptotic effect of butyrate and to avoid the confounding effects of
detachment-induced apoptosis (i.e., anoikis), we determined the
percentage of adherent cells exhibiting an apoptotic morphology. In the
early steps of butyrate-induced apoptosis, YAMC cells are still
attached to the culture flask. These cells lose their phospholipid membrane asymmetry and expose PS at the cell surface while maintaining their plasma membrane integrity. This process was monitored by using
annexin V-FITC. In addition, because the involvement of mitochondria in
apoptotic processes has been demonstrated (1, 19, 20),
butyrate-initiated dissipation of the

mt in adherent cells was
determined by laser cytometry. Although Heerdt et al. (19) have
demonstrated that butyrate-initiated dissipation of 
mt is required for
progression through the apoptotic cascade in nonadherent cells, our
novel observation that butyrate induces mitochondrial damage before
anoikis is consistent with the early involvement of mitochondria in
Fas-mediated apoptosis (36). Interestingly, mitochondrial damage
occurred after an initial lag period, paralleling the upregulation of
Fas-L expression. This is consistent with the idea that protein
synthesis or degradation is required for progression through the
apoptotic cascade. In keeping with this interpretation, there is
evidence that butyrate induction of apoptosis is dependent on the
inhibition of histone deacetylase and new protein synthesis (27). In
addition, with regard to the selective degradation and
posttranslational processing of functionally important proteins, the
ubiquitin pathway may play a role in the butyrate-induced apoptotic
program (36).
Butyrate is not a classical apoptogenic metabolite, capable of inducing
massive apoptosis. It is believed to serve as the primary energy source
for colonocytes in vivo (33, 45). It is therefore not altogether
surprising that the effect of butyrate on colonocyte apoptosis in
adherent cells is numerically small. In comparison, the total number of
cells (adherent + floating) undergoing apoptosis after butyrate
exposure is comparatively high, ~18% (Table 1). With regard to the
biological relevance of this effect, it is now clear that small changes
in apoptosis regulate crypt cell number (18), alter colon cancer risk
(4, 8), and modulate tumor behavior (39). In addition, the levels of
apoptosis in adherent-cell populations in our model system are similar
to reported levels in the human, rat, and mouse colon in vivo (4, 8,
14, 18). Therefore the YAMC cell culture system is a relevant model to
examine the mechanisms by which butyrate modulates colonic apoptosis.
In conclusion, butyrate stimulation of colonic apoptosis is mediated by
CD95 (APO-1/Fas). Our results also show that although butyrate is
capable of disrupting interactions between colonocytes and the
extracellular matrix, an anoikis-independent death pathway also exists.
Because Fas is downregulated or lost in the majority of colon
carcinomas, it remains to be determined whether butyrate treatment or
dietary fiber supplementation, will prove useful in the fight against
colorectal cancer.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Carl F. Ware for kindly providing recombinant Fas:Fc
and TNFR60:Fc and Dr. Robert Whitehead for supplying the YAMC cell
line. We also acknowledge the technical support of Dr. Laurie A. Davidson and Ms. Rong Cui.
 |
FOOTNOTES |
This work was supported in part by the Texas A&M Faculty Research
Development Program, Grants CA-59034 and CA-61750 from the National
Institutes of Health, and National Institute of Environmental Health
Sciences Grant P30-ES-09106.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. S. Chapkin,
442 Kleberg Center, Texas A&M Univ., College Station, TX 77843-2471 (E-mail: chapkin{at}acs.tamu.edu).
Received 4 February 1999; accepted in final form 19 April 1999.
 |
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