A diet high in dietary fibre and low in saturated fats is associated with a reduced
incidence of colorectal cancer (Sandler et al, 1993). The benefits of a high intake
of dietary fibre have been attributed to their fermentation in the gut to short-chain
fatty acids, including butyrate. Butyrate has been shown to increase apoptosis in
both colon adenoma and cancer cell lines in a p53-independent way (Hague et al, 1993),
an effect that is likely to contribute significantly to its protective effects. In
contrast, a diet high in saturated fats is associated with an increased level of faecal
secondary bile acids (Radley et al, 1993) and an increased risk of colorectal cancer
(Reddy and Wynder, 1977). Secondary bile acids, deoxycholic acid (DCA), ursodeoxycholic
acid (UDCA) and lithocholic acid, are produced by the actions of intestinal bacteria
on primary bile acids. Unconjugated bile acids have been shown to be tumour promoting
in rodent colonic epithelium (Narisawa et al, 1974; Mahmoud et al, 1999).
The majority of studies in the literature concerning the effects of butyrate and bile
acids on colonic epithelial cell proliferation and apoptosis have considered these
dietary factors individually. In vivo both are present in the colon and may influence
each other's actions directly or indirectly. For example, butyrate is known to modify
colonic pH and inhibit the colonic bacteria responsible for the production of bile
acids (MacDonald et al, 1978). We have shown previously that human adenoma cells,
AA/C1, treated with physiological levels of bile acids showed increased proliferation
(McMillan et al, 2000). Butyrate induced apoptosis in AA/C1 cells (Hague et al, 1993;
McMillan et al, 2000) and addition of bile acids to these cells reduced the level
of apoptosis, although this effect could be overcome by increasing the level of butyrate
(McMillan et al, 2000). Therefore, these two dietary factors interact at the cellular
level and the protective effect of butyrate may relate both to its apoptosis-inducing
effect per se and its ability to modify the antiapoptotic, proliferative effects of
secondary bile acids.
The molecular basis for the proapoptotic effects of butyrate have not been fully worked
out, although it does not involve induction of Fas expression or modulation of bcl-2
family protein expression (Bonnotte et al, 1998; Giardina et al, 1999). However, a
study by Medina et al (1997) has shown that butyrate induced activation of caspase
3 in colorectal cancer cells. Caspase 3 is a member of a cysteine protease family
that are involved specifically with the initiation and execution of the apoptotic
programme (reviewed in Kumar, 1995). Caspase 3 is a downstream effector caspase that
is activated by upstream caspases, predominantly caspases 8 and 9. Caspase 3 can be
activated by several routes, these include ligation of cell surface receptors, such
as Fas (Enari et al, 1996), which are associated with caspase 8; or by factors released
from mitochondria, that is, cytochrome c (Liu et al, 1996), in response to cellular
stress, which in turn activate caspase 9 (Liu et al, 1996). Caspase 3 can also be
activated by factors, including cellular stress, which leads to activation of the
p38 MAP (mitogen-activated protein) kinase pathway, independent of the mitochondria
(Shimizu et al, 1999).
The tumour-promoting effects of bile acids have been ascribed predominantly to their
potent activation of protein kinase C (PKC) (Huang et al, 1992). PKC is a family of
11 isoenzymes that are differentially regulated and play specific roles in the control
of cell proliferation, differentiation and apoptosis (Clemens and Trayner, 1992; Deacon
et al, 1997). With respect to apoptosis, PKC-α and -βll appear to be antiapoptotic
in most cells. PKC-α is inactivated by proapoptotic factors including ceramide (Lee
et al, 1996) and is known to phosphorylate bcl-2, potentiating its antiapoptotic function
in mitochondria (Ruvolo et al, 1998). PKC-βll is a mitotic lamin kinase (Goss et al,
1994) involved in the regulation of proliferation. In contrast, PKC-δ has a proapoptotic
function and can be activated proteolytically by caspase 3 (Ghayur et al, 1996). Furthermore,
transfection of cells with the caspase-3-generated fragment of PKC-δ is sufficient
to induce apoptosis, while the same fragment mutated to inactivate the kinase domain
is ineffective (Ghayur et al, 1996). In this study, we have considered the interaction
between bile acids and butyrate by investigating the signalling pathways activated
to effect their pro- and antiapoptotic actions.
MATERIALS AND METHODS
Cell culture
The colorectal adenoma cell line AA/C1 was provided by Professor C Paraskeva (Manning
et al, 1991) and was cultured as described previously (Williams et al, 1990). Prior
to treatment with bile acids or butyrate, cells were trypsinised and seeded at 106 cells
per flask, in triplicate, in DMEM (Gibco-BRL, Paisley, Scotland), containing 20% FCS
(Sera Laboratories International, Crawley, UK), 2 mM glutamine, 0.2 U ml−1 insulin,
1 μg ml−1 hydrocortisone sodium succinate (Sigma, Poole, UK), 100 U ml−1 penicillin
and 100 μg ml−1 streptomycin. After 3 days, the medium was replaced with a medium
containing 6 mM sodium butyrate or 10 μ
M UDCA (Sigma, UK). UDCA was added to the medium from a 10 mM stock in DMSO and solvent
controls were used throughout.
Assessment of PKC isoenzyme activation
PKC resides in the cytosol in its inactive state and translocates to the membrane
fraction upon activation. Following treatment with bile acid and butyrate, cells were
harvested by scraping, lysed and cytosol (soluble) and membrane (particulate) fractions
were isolated by centrifugation as previously described (Griffiths et al, 1996). Briefly
cell lysates were spun at 100 000 × g for 45 min at 4°C, the supernatant (soluble
fraction) was removed and the pellet (particulate fraction) was extracted in lysis
buffer (50 mM Tris-HCl, pH 7.4, 5 m, MgCl2, 1 mM EGTA, 1 m EDTA, 100 μg ml−1 leupeptin,
10 μg ml−1 pepstatin and 1 mM PMSF) containing 0.5% Triton X-100. Both fractions were
then taken up in SDS sample buffer and proteins were separated on 10% SDS–PAGE gels.
Protein content of each sample was determined prior to electrophoresis and 20 μg was
loaded per lane. Proteins were then transferred to PVDF membrane (Immobilon P, Millipore,
UK) and the PKC isoenzymes were detected by immunoblotting. Rabbit polyclonal antibodies
to PKC-α, -β (l and ll), -δ (Santa Cruz Biotechnology, US), -ɛ, -η and -ζ (Transduction
Laboratories, US) and HRP-conjugated anti-rabbit IgG antibodies (Amersham International
plc, UK) were used and blots were developed using enhanced chemiluminescence (ECL,
Amersham, UK). Equivalent loading was confirmed using an anti-β actin antibody (Sigma,
UK).
Measurement of p38 and p42/44 MAP kinase activation
Activation of p38 and p42/44 MAP kinases was determined by immunoblotting of whole
cell extracts using antibodies that detect the phosphorylated, active form of these
kinases (Upstate Biotechnology, US). Blots were developed using ECL. Equivalent loading
was confirmed using antibodies that detect total p38 and p42/44 MAP kinase.
Measurement of apoptosis
In studies to determine the effect of inhibitors of caspase 3, PKC isoenzymes and
MAP kinases on the regulation of apoptosis by bile acids and butyrate, inhibitors
were included in the culture medium with these agents and apoptosis was measured after
72 h. A volume of 0.1 μ
M Gö6976 (Calbiochem, UK) and 20 μ
M Rottlerin (Calbiochem, UK) were used to inhibit PKC-α and β and PKC-δ, respectively,
and 10 μ
M PD98059 and 1 μ
M SB202190 (Calbiochem, UK) were used as inhibitors of MEK 1, which lies upstream
of p42/44 MAP kinase and p38 MAP kinase, respectively. To confirm the involvement
of caspase 3 in butyrate-induced apoptosis, the caspase 3 inhibitor Ac-DEVD-fmk (Calbiochem,
UK) was used at 10 and 50 μ
M. The concentrations of inhibitors used were based on their IC50 values and our previous
experience with these agents (Pongracz et al, 1999; Cross et al, 2000). Apoptosis
was assessed by measuring annexin V binding and exclusion of propidium iodide, in
both attached and floating cells, using a commercial kit (Boehringer-Mannheim, Germany)
and FACS analysis.
Statistics
Data are presented as mean±standard deviation (s.d.) and means were compared by Student's
t-test. A value of P<0.05 was taken to indicate a significant difference between the
mean values.
RESULTS
Effect of sodium butyrate and UDCA on apoptosis of AA/C1 cells
The basal level of apoptotic cells detected in AA/C1 cultures was 12.75±0.8%. As shown
previously (Hague et al, 1993; McMillan et al, 2000), sodium butyrate (6 mM) induced
a significant increase in apoptosis in AA/C1 adenoma cells (P<0.005; Figure 1
Figure 1
Effect of butyrate and UDCA on apoptosis in AA/C1 cells. Levels of apoptotic cells
were determined in cultures of AA/C1 colon adenoma cells treated for 72 h with 6 mM
sodium butyrate alone, 10 μ
M sodium UDCA alone or butyrate and UDCA in combination. Apoptosis was assessed by
binding of annexin V and exclusion of propidium iodide. Results are mean ±s.d. (n=3)
and *denotes P<0.005.
). In contrast, 10 μ
M UDCA had no effect on spontaneous apoptosis of AA/C1, but did inhibit butyrate-induced
apoptosis (Figure 1).
Effect of sodium butyrate and UDCA on PKC isoenzyme activation
PKC-α, βl, βll and δ were detected in AA/C1 cells and were the predominant isoenzymes
expressed in these cells (Figure 2A
Figure 2
Effect of butyrate and UDCA treatment on PKC isoenzyme activation. PKC isoenzymes
(α, βl, βll and δ) were measured by Western blotting in soluble and particulate fractions
of (A) AA/C1 cells treated with or without 6 mM sodium butyrate for 2 h or (C) 10 μ
M UDCA for 24 h. (B) The 40 kDa fragment of PKC-δ was detected in whole cell lysates
of AA/C1 cells treated for 18 h with 6 mM sodium butyrate in the absence or presence
of the caspase 3 inhibitor Ac-DEVD-fmk. β-Actin was also measured as a loading control.
The estimated molecular weights on the immunoreactive bands in (A) are shown on the
right side of the figure. The blots shown are representative of three separate experiments
performed.
). PKC-ɛ, -η and -ζ were also detected, but were expressed at a low level and were
not affected by either bile acid or butyrate treatments (data not shown). Treatment
of AA/C1 cells (Figure 2A) with 6 mM sodium butyrate induced a modest translocation
of full-length 78 kDa PKC-δ from the soluble to the particulate fraction, 2 h after
addition of butyrate. Densitometric analysis of blots from three separate experiments
showed that the percentage of total PKC-δ present in the particulate fraction increased
from 52.4±4% in untreated cells to 88.3±9% in butyrate-treated cells. Translocation
of PKC-α, -βl and -βll was not detected in response to butyrate treatment (Figure
2A), even up to 24 h after addition of butyrate (data not shown). Further studies
revealed that a 40 kDa protein was detected by the anti-PKC-δ antibody in butyrate-treated
cells. This fragment was not detected until 18–24 h of butyrate treatment and after
the translocation of full-length PKC-δ (Figure 2B). PKC-δ can be proteolytically activated
by caspase 3, leading to the generation of a constitutively active 40 kDa fragment
(Ghayur et al, 1996). The appearance of the 40 kDa PKC-δ fragment was inhibited by
inclusion of the caspase 3 inhibitor Ac-DEVD-fmk in butyrate-treated cultures (Figure
2B), suggesting that this represented the caspase activated form of PKC-δ.
Treatment of AA/C1 cells with 10 μ
M UDCA induced translocation of PKC-α to the particulate fraction after 24 h, with
no consistent change detected for the other PKC isoenzymes (Figure 2C). Densitometric
analysis of three blots showed that the fraction of PKC-α associated with the particulate
fraction increased from 27.4±4% in untreated to 59.8±7% in UDCA-treated cells. Thus,
PKC isoenzymes were activated differentially by butyrate and UDCA.
Effect of butyrate and UDCA on p38 and p42/44 MAP kinase activation
Butyrate (6 mM) induced activation of p38 MAP kinase in AA/C1 cells, indicated by
an increase in the level of phosphorylated p38 MAP kinase, but did not induce activation
of p42/44 MAP kinase (Figure 3
Figure 3
Effect of butyrate and UDCA on p38 and p42/44 MAP kinase activity. AA/C1 cells treated
with 6 mM butyrate or 10 μ
M UDCA for 24 h. Cells were examined for the presence of activated, phosphorylated
p38 MAP kinase (pP38) and p42/44 MAP kinase (pP42/44) by Western blotting. PD98059
was also included with UDCA treatments (lane 5) to inhibit activation of p42/44 MAP
kinase by MEK1, and SB202190 was included with butyrate treatments (lane 3) to inhibit
p38 MAP kinase. Equal loading of gels was confirmed using an antibody to total P38
and P42/44 MAP kinase. The blot shown is a representative of three similar experiments
performed.
). In contrast, active p42/44 MAP kinase was increased in cultures of AA/C1 treated
with UDCA (Figure 3) and 10 μ
M UDCA did not increase levels of activated p38 MAP kinase (Figure 3). Therefore,
butyrate activated p38 MAP kinase, which has been shown to be involved in pathways
leading to apoptosis and UDCA activated the p42/44 MAP kinase pathway, which has been
implicated in cell proliferation and survival.
Effect of PKC, MAP kinase and caspase 3 inhibitors on the actions of butyrate and
UDCA
To determine whether the signals through the PKC and MAP kinase pathways were required
for the effects of butyrate and UDCA, inhibitors of these pathways were used. The
PKC-δ inhibitor Rottlerin and the p38 MAP kinase inhibitor SB202190 blocked apoptosis
induced by butyrate (Figure 4A
Figure 4
Effect of PKC, MAP kinase and caspase 3 inhibitors on the effects of UDCA and butyrate.
(A) AA/C1 cells were incubated with 6 mM butyrate alone and in the presence of 10 μ
M PD98059, 1 μ
M SB202190, 20 μ
M Rottlerin or 20 μ
M Ac-DEVD-fmk. Apoptosis was measured after 72 h by annexin V binding and exclusion
of propidium iodide. Values for butyrate treatment were compared with butyrate combined
with the inhibitors used. (B) AA/C1 cells were incubated with 10 μ
M UDCA in the absence or presence of 10 μ
M PD98059, 1 μ
M SB202190 or 0.1 μ
M Go6976. Attached cell number was measured after 96 h and expressed as a percentage
of the value for untreated cells. Data are mean ±s.d. of three separate experiments
and *denotes P<0.05 and **denotes P<0.01.
). As PKC-δ was also activated via caspase 3, the effects of the inhibitor Ac-DEVD-fmk
were determined. Ac-DEVD-fmk also reduced apoptosis in butyrate-treated cultures (Figure
4A). The MEK 1 inhibitor PD98059 inhibits the activation of p42/44 MAP kinase indirectly
and did not inhibit the proapoptotic actions of butyrate (Figure 4A). PD98059 blocked
the effect of UDCA, inhibiting the increase in cell number seen with this bile acid
(Figure 4B). The proliferative effect of UDCA was confirmed by measuring thymidine
incorporation, which showed that incorporation of tritiated thymidine increased from
48.9±0.9 to 60.2±2.1 × 103 d.p.m. per 105 cells after 96 h treatment. The proliferative
effects of UDCA were also reduced by Gö6976 (Figure 4B), an inhibitor of PKC-α and
-β that does not affect PKC-δ (Gschwendt et al, 1996), confirming that both PKC and
p42/44 MAP kinase are involved in mediating the effects of UDCA. The p38 MAP kinase
inhibitor SB202190 had no effect on the proliferative effects of UDCA (Figure 4B).
The inhibitor studies thus confirmed that modulation of PKC and MAP kinase signalling
pathways by butyrate and UDCA was involved in mediating their effects on colorectal
adenoma cell apoptosis and proliferation.
DISCUSSION
In all tissues, including the colon, cell number and phenotype are maintained by a
correct balance between cell proliferation, differentiation and apoptosis. Disruption
of this balance underlies the development of cancer (Williams, 1991). In the colonic
crypt, stem cells are located at the base and as they differentiate they progress
up the crypt, eventually dying by apoptosis and are sloughed off into the colonic
lumen (Potten and Grant, 1998). The high incidence of colorectal cancer in Western
society has been attributed to a diet high in saturated fat and low in dietary fibre
(Sandler et al, 1993). A high intake of saturated fat results in increased production
of bile acids and raised levels of secondary bile acids, produced in the colon (Hill,
1986), have been measured in patients with adenomatous polyps and colorectal cancer
(Radley et al, 1993). While bile acids are not mutagenic, they have been shown to
act as tumour promoters in animal studies (Narisawa et al, 1974; Mahmoud et al, 1999)
and PKC has been identified as their molecular target (Pongracz et al, 1995). We confirm
here that the secondary bile acid UDCA was able to activate PKC in whole cells (Huang
et al, 1992) and show that this action was restricted to the classical PKC isoenzyme
PKC-α. Although UDCA was the focus of this study, we have also shown that other bile
acids can inhibit butyrate-induced apoptosis, including CDCA (McMillan et al, 2000),
and this bile acid also activates PKC-α (data not shown). Interestingly, the literature
suggests that PKC-α has a predominantly antiapoptotic role (Deacon et al, 1997). For
example, PKC-α is inhibited during ceramide-induced apoptosis (Lee et al, 1996) and
expression of a dominant-negative PKC-α induced apoptosis in CHO cells (Whelan and
Parker, 1998). Thus, those bile acids known to promote cell proliferation and inhibit
butyrate-induced apoptosis, that is, UDCA, CDCA and DCA (Mahmoud et al, 1999; McMillan
et al, 2000), are likely to be mediated by the activation of PKC-α. This proposal
is supported by the ability of the PKC inhibitor Gö6976 to block the proliferative
effects of UDCA on AA/C1 cells.
Our studies also revealed that a kinase lying downstream of PKC-α, namely p42/44 MAP
kinase, was also activated following UDCA treatment. This kinase is the original member
of the mitogen-activated protein kinase family (MAP kinases) that are activated by
a variety of mitogens, including growth factors and the PKC activator TPA (Kyriakis
et al, 1994). PKC-α is known to phosphorylate and activate Raf-1, a serine threonine
kinase that activates the kinase upstream of p42/44 MAP kinase, MEK1 (Kolch et al,
1993). Inhibition of MEK1 was able to block the effects of UDCA, confirming that the
PKC-α and p42/44 MAP kinase play a role in mediating the effects of bile acids on
AA/C1 cells.
In contrast to the activation of survival/proliferative pathways by UDCA, butyrate
was found to activate pathways known to mediate cell death by apoptosis, namely PKC-δ
and p38 MAP kinase, consistent with the induction of apoptosis by this agent (Hague
et al, 1993; McMillan et al, 2000). Butyrate has also been shown to activate p38 MAP
kinase in Caco-2 cells (Ding et al, 2001). Loss of PKC-δ protein expression has been
reported in human adenocarcinoma tissue (Craven and deRubertis, 1994) and this may
underlie the reduced responsiveness of cancer tissue to the apoptosis-inducing effects
of butyrate (Bonnotte et al, 1998). Butyrate has been shown previously to activate
NF-κB in a PKC-dependent manner, although the PKC isoenzyme involved was not determined
(Giardina et al, 1999). A signalling pathway involving both p38 MAP kinase and NF-κB
and leading to activation of caspase 3 has been described (Ichijo et al, 1999; Shimizu
et al, 1999). Our data suggest therefore that activation of full-length PKC-δ, which
occurred before caspase 3 activation of PKC, may initiate the signals leading to p38
MAP kinase and NF-κB activation. The latter will then result in caspase 3 activation
and further activation of PKC-δ by caspase-3-mediated proteolysis. Although not investigated
here, butyrate is also known to induce caspase 3 activation via the mitochondrial
route. The latter involves generation of reactive oxygen species and release of cytochrome
c from the mitochondria. Thus, caspase 3 activation by butyrate is achieved by two
pathways and will ensure that cleavage and activation of the proapoptotic PKC-δ is
achieved.
The targets of PKC-δ that effect its involvement in apoptosis have not been fully
described, but appear to include predominantly nuclear proteins. The caspase-3-activated
form of PKC-δ has been shown to inhibit DNA-PK, a nuclear protein involved in DNA
repair (Bharti et al, 1998) and to phosphorylate nuclear lamin B prior to disassembly
of the nuclear lamina (Cross et al, 2000). Although translocation of PKC-δ was shown
here, the particular membrane involved was not identified. However, we have shown
previously that PKC-δ translocated to the nuclear membrane during apoptosis in T cells,
HL60 cells and neutrophils (Pongracz et al, 1999; Scheel-Toellner et al, 1999; Cross
et al, 2000).
In summary, we have shown that the opposing effects of bile acid and butyrate, used
at concentrations within the physiological range, on colon adenoma cell apoptosis
are mediated via differential activation of signalling pathways that regulate apoptosis.
The ultimate level of apoptosis in the colon may therefore be dictated by the balance
of signals through the pro- and antiapoptotic PKC isoenzymes and MAP kinases. Such
a proposal is supported by the data of Ding et al (2001), which showed that butyrate-induced
apoptosis of Caco-2 cells was potentiated by the MEK inhibitor PD98059 and involved
activation of p38 MAP kinase as reported here. Any beneficial effects of butyrate
with regard to colon cancer will only be realised if levels of this agent are high
enough in the colon to overcome the tumour-promoting signals induced by unconjugated
bile acids.
Future studies will determine whether the signalling pathways activated by bile acids
are able to inhibit directly the activation of proapoptotic signalling pathways, PKC-δ
and p38 MAP kinase, activated by butyrate, or whether they act downstream of these
signals, for example by inducing the phosphorylation and activation of bcl-2 (Ruvolo
et al, 1998).