Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215)

Abstract

The effects of short chain fatty acids on a colon carcinoma cell line, LIM1215, have been studied. Of the four short chain fatty acids tested only butyrate at 1 mmol/l and 10 mmol/l and acetate at 10 mmol/l had significant effects on this cell line. The addition of butyrate to growth medium affected the growth rate and the production of alkaline phosphatase, dipeptidyl peptidase IV and carcinoembryonic antigen. Butyrate at a final concentration of 1 mmol/l increased the doubling time of the cells from 26 hours to 72 hours and decreased the cloning efficiency of the cells from 1.1% to 0.054%. Alkaline phosphatase concentrations increased rapidly in cells cultured in 1 mmol/l butyrate reaching peak levels after four days with alkaline phosphatase concentrations increasing more than six-fold. Levels of dipeptidyl peptidase IV and carcinoembryonic antigen were also increased after culture in butyrate containing medium. The number of alkaline phosphatase containing and dipeptidyl peptidase IV containing cells increased markedly in butyrate containing cultures. In contrast the number of mucus containing cells decreased in cultures grown in medium containing butyrate. This differentiating effect of butyrate on colon carcinoma cells may be relevant to the presence of butyrate in the colonic contents and the relationship between short chain fatty acids and fibre intake.

Full text

Gut,
1986,
27,
1457-1463
Effects
of
short
chain
fatty
acids
on
a
new
human
colon
carcinoma
cell
line
(LIM1215)
R
H
WHITEHEAD,
G
P
YOUNG,
AND
P
S
BHATHAL
From
the
Melbourne
Tumour
Biology
Branch,
Ludwig
Institute
for
Cancer
Research,
PO
Royal
Melbourne
Hospital,
Melbourne;
University
of
Anatomical
Pathology,
The
Royal
Melbourne
Hospital,
Victoria,
Australia
SUMMARY
The
effects
of
short
chain
fatty
acids
on
a
colon
carcinoma
cell
line,
LIM1215,
have
been
studied.
Of
the
four
short
chain
fatty
acids
tested
only
butyrate
at
1
mmol/l
and
10
mmol/l
and
acetate
at
10
mmol/l
had
significant
effects
on
this
cell
line.
The
addition
of
butyrate
to
growth
medium
affected
the
growth
rate
and
the
production
of
alkaline
phosphatase,
dipeptidyl
peptidase
IV
and
carcinoembryonic
antigen.
Butyrate
at
a
final
concentration
of
1
mmol/l
increased
the
doubling
time
of
the
cells
from
26
hours
to
72
hours
and
decreased
the
cloning
efficiency
of
the
cells
from
1-1%
to
0(054%/o.
Alkaline
phosphatase
concentrations
increased
rapidly
in
cells
cultured
in
mmol/l
butyrate
reaching
peak
levels
after
four
days
with
alkaline
phosphatase
concentrations
increasing
more
than
six-fold.
Levels
of
dipeptidyl
peptidase
IV
and
carcinoembryonic
antigen
were
also
increased
after
culture
in
butyrate
containing
medium.
The
number
of
alkaline
phosphatase
containing
and
dipeptidyl
peptidase
IV
containing
cells
increased
markedly
in
butyrate
containing
cultures.
In
contrast
the
number
of
mucus
containing
cells
decreased
in
cultures
grown
in
medium
containing
butyrate.
This
differentiating
effect
of
butyrate
on
colon
carcinoma
cells
may
be
relevant
to
the
presence
of
butyrate
in
the
colonic
contents
and
the
relationship
between
short
chain
fatty
acids
and
fibre
intake.
We
have
recently
established
a
new
colon
carcinoma
cell
line
with
the
capacity
to
spontaneously
dif-
ferentiate
to
some
degree.
'
Electron
microscopic
studies
show
the
presence
of
two
cell
types,
one
with
organised
areas
of
microvilli
and
the
other
with
mucin
inclusions.
In
view
of
the
presence
of
a
range
of
fatty
acids
in
the
colonic
lumen,
this
study
has
set
out
to
test
the
effects
of
various
short
and
medium
chain
fatty
acids
on
the
growth
and
differentiation
of
this
cell
line.
Cellular
differentiation
has
been
followed
by
monitoring
alkaline
phosphatase,
dipeptidyl
peptidase
IV,
carcinoembryonic
antigen
concentrations
and
mucin
staining.
Sodium
butyrate
has
been
shown
to
induce
differentiation
in
various
cell
types
in
vitro
-
for
example,
colon
carcinoma,2
Syrian
hamster
cells3
and
erythroleukaemia
cells.4
The
effect
of
butyrate
in
vivo
is
unknown
as
it
is
not
feasible
to
administer
Addrcss
for
correspondcnce:
Dr
R
H
Whitehead,
cell
Biology
Laboratory.
Ludwig
Institute
for
Cancer
Research,
Royal
Melbournc
Hospital.
Victori
3050.
Australia.
Reccived
for
publication
22
April
1986
butyrate
systemically
because
it
and
other
short
chain
fatty
acids
such
as
acetate
and
propionate
are
rapidly
cleared
from
the
circulation.
The
effects
of
acetate
or
propionate
on
cell
differentiation
is
uncertain.
Butyrate,
acetate,
and
propionate
are
normally
present
in
the
lumen
of
the
large
bowel;6
an
important
consideration
as
the
large
bowel
is
a
common
site
of
cancer.
These
three
short
chain
fatty
acids
comprise
the
major
solute
fraction
of
faecal
water
(190
mmol/16)
being
produced
by
fermentation
of
malabsorbed
or
non-absorbed
dietary
carbohy-
drates
-
for
example,
fibre,
by
anaerobic
bacteria
normally
resident
in
the
large
bowel.6
These
short
chain
fatty
acids
are
readily
taken
up
by
colonic
mucosa
and
are
preferred
to
glutamine
and
glucose
as
respiratory
fuels
for
the
colonocyte.7
In
this
natural
setting,
short
chain
fatty
acids
have
the
potential
to
modify
growth
of
colonic
neoplasms
and
perhaps
even
to
protect
against
their
genesis.
The
modulation
of
cell
differentiation
by
means
such
as
these
has
potential
in
the
management
and
preven-
tion
of
colon
carcinoma,
because
the
amounts
of
1457
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

Whiteliealc,
Yolu'g,
anld
Bliathal
short
chain
fatty
acids
in
the
colon
can
be
modified
by
dietary
means.
Methods
CELL
LINE
The
LIM1215
cell
line
has
been
described
in
full
elsewhere.'
In
brief,
the
cell
line
was
obtained
from
a
34
year
old
man
with
a
family
history
of
colorectal
cancer.
His
father
and
two
brothers
had
previously
developed
colorectal
cancer.
At
laparotomy,
the
patient
was
found
to
have
widespread
abdominal
metastases
and
the
primary
lesion
in
the
ascending
colon
was
judged
to
be
inoperable.
The
cell
line
was
derived
from
tissue
taken
from
involved
omentum.'
Cells
were
maintained
in
RPMI
1640
medium
supplemented
with
10%
fetal
calf
serum,
100
nmol/l
insulin,
2
[tmol/l
hydrocortisone,
10
[tmol/l
a-thioglycerol,
50
U/ml
penicillin
and
50
p.g/ml
streptomycin
as
described
previously.8
The
cells
were
passaged
by
trypsinising
with
a
solution
of
04
I%
trypsin
and
0-05
mmol/l
ethylene
diamine
tetraacetic
acid
(EDTA)
in
phosphate
buffered
saline
(pH
7.2).
GROWTH
STU1)IES
All
growth
studies
were
donle
in
the
growth
medium
described
above.
Cells
were
trypsinised
and
resus-
pended
in
medium
at
a
concentration
of
l()
cells/ml.
One
millilitre
aliquots
of
the
cell
suspensioni
were
then
dispersed
into
each
well
of
24
well
plates
(Nunc,
Denmark).
Appropriate
dilutions
of
aqueous
solutions
of
short
chain
fatty
acids
[all
fatty
acids
tested
were
obtained
from
Sigma
Chemical
Co,
St
Louis,
Mo]
were
then
added
to
triplicate
wells
and
the
plates
incubated
at
37°C
in
an
atmosphere
of
5%
CO2
in
air
for
up
to
seven
days.
After
incubation,
the
medium
from
each
well
was
removed
and
stored
in
numbered
tubes.
One
millilitre
trypsin-EDTA
solution
was
added
to
each
well
and
the
plate
incubated
until
all
cells
had
been
detached
from
the
growth
surface.
The
cell
suspen-
sion
in
each
well
was
then
added
to
the
correspond-
ing
medium
and
the
total
cell
count
per
well
determined
using
a
haemocytometer.
Viability
was
determined
by
dye
exclusion.
ClONING
STUDIES
Monolayer
cultures
were
trypsinised
and
the
cells
suspended
in
growth
medium.
The
suspension
was
filtered
through
sterile
nylon
gauze
(50
,t
mesh)
and
then
through
sterile
cotton
wool
to
remove
clumps
and
the
resulting
cell
suspension
checked
for
clumps
by
microscopy.
The
cell
suspension
was
then
counted
using
at
haemocytometer
and
the
cell
count
adjusted
to
give
cell
concentrations
ranging
from
102/ml
to
l('/ml
and
plated
in
duplicate
in
2
ml
volumes
in
35
mm
dishes.
The
colonies
formed
were
counted
at
10
days.
Aggregates
of
more
than
50
densely
packed
cells
were
counted
as
colonies.
Al
KAl.INE
P'HOSIPHATASE
Cells
(2x
l('/ml)
were
grown
in
flasks
in
media
containing
short
chain
fatty
acids.
After
incubation,
the
culture
medium
was
collected
and
stored.
The
cell
monolayer
was
then
rinsed
twice
in
1i)
mmol/l
Tris
HCl
buffer.
pH
7.4,
containing
250
mmol/l
mannitol
buffer
and
in
the
same
buffer
containing
50
mmol/l
mannitol.
Trypsin
was
not
used.
The
cells
were
removed
by
scraping
in
ice
cold
50
mmolIl
mannitol
buffer,
centrifuged
and
the
cell
pellet
stored
at
-20°C
until
assayed.
Immediately
before
assay,
the
cell
pellet
was
homogenised
for
15
seconds
using
a
size
C
tissue
grinder
(A
H
Thomas,
Philadelphia,
PA)
with
a
Teflon
pestle
rotating
at
full
speed.
For
alkaline
phosphatase
and
protein
assays,
aliquots
of
homogenate
were
made
up
to
0-1%
Triton
X-100
(Sigma
Chemical
Co,
St
Louis)
before
assay.
Determination
of
alkaline
phosphatase
activ-
ity
was
carried
out
with
p-nitrophenyl
phosphate
as
substrate
according
to
the
method
previously
described'
using
a
2-amino-2-methylpropanol
(Sigma)
buffer.
Protein
was
measured
with
bovine
gamma-globulin
as
standard."'
All
determinations
were
made
in
duplicate
or
triplicate.
CARCINOEMBRYONIC
ANTIGEN
Carcinoembryonic
antigen
(CEA)
was
measured
using
a
solid
phase
radioimmunoassay
kit
(Abbot
CEA-RIA
Diagnostic
kit
no.
7875,
Abbot
Labor-
atories,
North
Chicago).
Carcinoembryonic
antigen
release
into
culture
media
was
measured
by
direct
assay
of
media
diluted
in
CEA
extraction
buffer.
Membrane
bound
CEA
was
measured
in
homogen-
ates
prepared
as
above,
solubilised
by
1%
Nonidet-
P40
(BDH
Chemical
Ltd,
Poole,
England)
at
4°C
for
30
minutes
and
then
centrifuged
at
low
speed
(5000
g
for
10
minutes)
to
collect
solubilised
material.
[Triton
X-100
did
not
achieve
adequate
solubilisation
of
CEA.]
Carcinoembryonic
antigen
concentrations
were
expressed
as
ngm/mg
cell
protein.
SCANNING
ELECTRON
MICROSCOPY
STUI)IES
For
scanning
electron
microscopy,
the
cells
were
grown
for
five
days
on
plastic
coverslips
(Therma-
nox,
Lux
Scientific
Corp,
Newbury
Park,
CA),
washed
briefly
in
PBS,
fixed
in
2-5%
glutiaraldehyde
in
0
1
M
cacodylate
buffer
for
30
minutes,
washed
in
three
changes
of
cacodylate
buffer
containing
0(25
iTIol/l
sucrose,
postfixed
in
27%,
osmium
tetroxide
for
1458
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

Effects
of
short
chain
fatty
acids
on
a
new
humcan
colon
carcinoma
cell
line
(LIMI
215)
one
hour,
washed
again
with
buffer,
dehydrated
through
a
graded
series
of
ethanol
and
dried
by
the
critical
point
method
using
liquid
carbon
dioxide
as
the
infiltrating
medium.
The
coverslips
were
then
mounted
on
stubs,
shadow-coated
with
gold
and
viewed
in
an
Etec
Autoscan
at
20
kV.
DIPEPTIDYI
PEPTIDASE
IV
(DPPIV)
This
enzyme
was
assayed
using
glycyl-L-proline-p-
nitroanilidine
as
substrate
as
described
by
Nagatsu
et
al."
IMMUNOCHEMICAL
STAINING
LIM1215
cells
were
grown
on
8-chamber
Lab
Tek
slides
(Miles
Laboratories
Inc,
Naperville,
USA)
for
five
days
either
in
control
medium
or
in
medium
containing
1
mmol/l
sodium
butyrate.
After
five
days,
the
slides
were
washed
in
phosphate
buffered
saline,
fixed
in
acetone
at
-20(C
for
10
minutes
and
air
dried.
Alkaline
phosphatase
activity
was
demon-
strated
histochemically
using
the
Napthol
AS-BI
(Sigma)
method.'2
Mucus
was
demonstrated
using
two
monoclonal
antibodies
raised
against
either
human
colonic
mucosal
membranes
(MON024,
Gardner
I,
in
preparation)
or
purified
colonic
mucous
(LIMA,
kindly
supplied
by
J
Ma,
Monash
University
Melbourne).'3
Dipeptidyl
peptidase
IV
(DPPIV)
was
demonstrated
using
a
monoclonal
antibody
kindly
supplied
by
Dr
H-P
Hauri,
Basel,
Switzerland.
14
STATISTICAL
ANALYSIS
All
statistical
comparisons
were
made
using
the
Wilcoxon's
rank
sum
test.
Results
GROWTH
STUDIES
The
effects
of
fatty
acids
at
a
range
in
concentrations
on
the
growth
of
LIM1215
cells
in
monolayer
culture
are
summarised
in
Table
1,
where
growth
is
expressed
as
a
percentage
of
control
growth.
Three
of
the
short
chain
fatty
acids
tested
were
tested
at
three
concentrations
and
of
these
only
propionate
at
10
mmol/l
and
butyrate
at
1
0
mmol/l
and
10
mmol/l
significantly
suppressed
growth
(p<0.05).
Valerate
and
isovalerate
were
only
tested
at
a
single
concen-
tration
(1
mmol/l)
but
neither
suppressed
the
growth
of
LIM1215
cells
at
this
concentration
(results
not
shown).
There
was
no
significant
stimulation
of
growth
with
any
of
the
fatty
acids
tested.
Because
of
its
effects,
butyrate
was
studied
further.
Growth
curve
studies
(Fig.
1)
indicated
that
1
mmol/l
butyrate
increased
the
doubling
time
from
26
hours
in
control
cultures
to
72
hours
in
butyrate
treated
cultures.
The
total
cell
yield
was
also
Table
1
Growuhi
of
LIM1215
cells
in
tile
presence
of
s/tort
chaini
fiattY'
acids
(e/lt
r
vei,
l'
(
x
1(0
)
(ii)
Acctatc
(I
m1ilol
8X±7+()8
1-0
nmilol/l
7-4+1-1
10(0
11mrnol/I
5-9+0()5
Propionatc
()-
Itimilol/l
7
5+1
3
1-(0
niiloUl
6X8+3()3
10-0
iminiol/l
I
3+()_-92
Butyratc
0(
mm1111oIl
68+81
4
1
)
nimol/I
20
()+±()
2
1C
(
milloul
1-4+()
1,
Control
8-11
±12
"Mean
of
threc
cuLltuircs
+
I
rcduction
in
growth
(p<()-()5).
65
-
1-
o
60-
a
55-
5-0
st.and.ard
devviation.
Significalit
-,3
3-_
I-
//
X-
--
-
.
/
8
0
2
4
6
Incubation
period
(days)
Fig.
Growth
of
LIM1215
cells
in
either
control
or
butvrate-containing
(I
mmnolll)
mnedia.
SEM
inidicated
bY
vertical
bars.
(N=3
for
each
data
poimit).
decreased.
This
effect
was
not
caused
by
toxicity,
however,
as
the
viability
of
the
cells
was
not
affected
and
dead
cells
were
not
shed
into
the
medium.
Growth
in
butyrate
containing
medium
also
in-
duced
a
change
in
the
morphology
of
the
cells.
The
cells
became
much
larger
and
flatter
within
two
to
three
days
of
the
addition
of
butyrate
to
the
culture.
This
was
shown
by
demonstrating
an
increase
in
low
angle
scatter
(a
parameter
related
to
cell
size)
using
a
cytofluorograph.
The
mean
channel
for
low
angle
scatter
for
control
cells
was
73
and
this
increased
to
95
for
cells
grown
in
butyrate
containing
medium
for
5
days.
CLONING
EFFICIENCY
LIM1215
cells
were
cloned
at
low
cell
density
in
1459
Control
Butyrate
1
mmi
/
..
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

Whitehead,
Young,
and
Bhathal
liquid
culture
either
in
control
medium
or
medium
containing
1
mmol/l
sodium
butyrate.
In
control
cultures,
11+1
colonies
were
obtained
with
an
inoculum
of
103
cells/ml
giving
a
cloning
efficiency
of
1*1%.
In
contrast,
in
butyrate
containing
cul-
tures,
5±3
colonies
were
obtained
with
a
cell
inoculum
of
104/ml
giving
a
cloning
efficiency
of
0O05%
and
maximal
cloning
was
only
obtained
at
an
inoculum
of
105
cells/ml.
ALKALINE
PHOSPHATASE
Alkaline
phosphatase
specific
activity
was
signifi-
cantly
increased
(p<005)
by
the
addition
of
acetate
(1-0
mmol/l
and
10-0
mmol/l),
propionate
(1-0
mmol/l)
and
most
markedly
by
butyrate
at
a
concentration
of
1-0
mmol/l
(Fig.
2).
The
effects
of
various
concentrations
of
butyrate
on
cell
growth
and
alkaline
phosphatase
specific
activity
are
shown
in
Figure
3.
A
concentration
of
1
mmol/l
significant-
ly
(p<002)
suppressed
growth
and
was
associated
with
a
marked
increase
(p<002)
in
alkaline
phos-
phatase
activity.
Figure
4
shows
that
the
butyrate
induced
increase
in
alkaline
phosphatase
specific
activity
appeared
within
24
hours
but
plateaued
at
three
to
four
days.
The
increase
had
become
significant
by
day
two
(p<002).
In
contrast,
total
activity
continued
to
increase
progressively
relative
to
controls
(Fig.
5).
ULTRASTRUCTURE
STUDIES
Scanning
electron
microscope
studies
have
con-
firmed
the
morphological
observations
on
changes
in
cell
size
in
butyrate
containing
medium.
Interes-
tingly,
the
distribution
and
number
of
microvilli
did
600
-
1l/
500
-
0
3
°
400
lO
i300
200-
T
z100
Fig.
2
Influence
of
various
short
chain
fatty
acids
on
alkaline
phosphatase
activity.
Fatty
acids
added
were
acetic,
propionic
(propion),
butyric,
valeric
and
isovaleric
(isovaler).
Cultures
were
maintained
for
five
days
before
the
enzyme
was
assayed.
SEM
indicated
by
vertical
bars.
(n
=3)
0
01
10
10
Sodium
butyrate
(mmol
/
I)
Fig.
3
Efect
of
various
concentrations
of
butyrate
on
cell
growth
and
alkaline
phosphatase
activity.
SEM
indicated
by
vertical
bars.
(n=3)
100-
u
,E
E
0
0.
0-
80
60
40
20
0
leo
"""
I
IButyrate
lmmol/l
----
I
Control
yI
I
0
2
4
6
Incubation
period
(
day
)
Fig.
4
Time
course
of
induction
of
alkaline
phosphatase
activity
(mUlmg
cell
protein)
when
cultured
in
I
mmolll
butyrate.
SEM
indicated
by
vertical
bars.
(n
=3)
800
700
2~
600
400
300
)
200
t
100
-!
<
o
T--Tl\
.0
~
~ ~ ~ ~ ~
/I|
Alk.
phosphotase
H--
*
|CEA
0__
-
g
0
2
4
6
Incubation
period
(
days)
Fig.
5
Time
course
of
total
alkaline
phosphatase
activity
and
carcinoembryonic
antigen
per
culture,
when
cultured
in
I
mmolll
butyrate.
SEM
indicated
by
vertical
bars.
(n=3)
8
-400
m
-300
>
0
0
.200
:3
a3
*0
8
1460
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

Effects
of
short
chain
fatty
acids
on
a
new
human
colon
carcinoma
cell
line
(LIM1215)
not
appear
to
be
altered
by
growth
in
butyrate
containing
medium
(Figs
6a
and
6b).
CEA
PRODUCTION
Monolayers
cultured
in
the
presence
of
butyrate
produced
significantly
more
CEA
than
did
control
cultures.
Carcinoembryonic
antigen
production
con-
tinued
to
increase
with
length
of
exposure,
although
Fig.
6
Scanning
electron
micrographs
of:
(a)
culture
grown
in
control
medium;
and
(b)
cultures
grown
in
medium
containing
1
mmol/l
butyrate.
Both
cultures
were
grown
for
five
days
before processing.
There
were
no
differences
in
either
the
density
or
the
distribution
of
microvilli.
Table
2
Effects
of
I
mmolll
butvrate
oni
antigen
expression
by
LIM1215
cells
in
monolayer
culture
Control
Buivsrate
(I
nitiolll)
Alkaline
phosphatase
1%'Y
+t
30%
t
DPPIV
3%
+
60%
+
Mucus-
Mono24
25%
+
5%
+
LIMA
20%
+
+
I
(%
+
*Percentage
of
cells
positive
in
five
day
cultures.
tintcnsity
assessed
on
a
scale
varying
from
+
=barely
discernablc
to
t=extremely
strong.
not
as
rapidly
as
for
alkaline
phosphatase
(Fig.
5).
Total
CEA
production
per
culture
flask
increased
three
to
four-fold
(p<0-05)
during
the
first
four
days
of
culture
(Fig.
5),
but
then
fell
back
towards
control
values.
In
contrast,
alkaline
phosphatase
concentra-
tions
continued
to
rise.
DIPEPTIDYLAMINOPEPTI
DASE
Butyrate,
1
mmol/l,
increased
activity
of
this
enzyme
approximately
350/
from
4-58+0-49
mU/mg
protein
(x±SE)
in
control
media
to
6-30±125
mU/mg
in
butyrate
containing
media,
after
five
days
of
culture.
HISTOCHEMICAL
STUDIES
The
percentage
of
cells
expressing
alkaline
phospha-
tase
activity
or
containing
dipeptidyl
peptidase
IV
was
increased
markedly
by
culture
in
1
mmol/l
butyrate
(Table
2).
The
intensity
of
the
histo-
chemical
reaction
for
alkaline
phosphatase
was
also
increased.
In
contrast,
the
percentage
of
cells
in
the
culture
staining
with
antimucus
antibodies
de-
creased
in
butyrate
cultures
(Table
2).
Discussion
Butyrate
is
a
potent
differentiating
agent
in
many
systems.3
15
16
It
has
been
widely
studied
in
a
murine
erythroleukaemia
system'7-19
and
has
been
shown
to
induce
granulocyte
differentiation
in
human
pro-
myelocytic
leukaemia
cells.20
Butyrate
also
has
differentiating
effects
on
human
retinoblastoma
cells
in
monolayer
culture2'
and
on
antigen
produc-
tion
by
the
human
breast
carcinoma
cell
line
MCF-
7.5
Although
the
mode
of
action
of
butyrate
is
still
unclear,
it
is
known
that
butyrate
alters
histone
acetylation
patterns
by
reducing
nuclear
histone
acet
ltransferase
and
inhibiting
histone
deactyl-
ase.
6
22
Butyrate
also
inhibits
phosphorylation
of
histones
and
can
interfere
with
methylation
of
nuclear
protein
fractions.'2
23
Although
butyrate
has
been
studied
in
many
systems
it
is
of
interest
in
relation
to
the
colon
as
it
is
present
in
high
concentration
in
colonic
contents.6
Other
short
1461
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

1462
Whitehead,
Young,
and
Bhathal
chain
fatty
acids
are
also
present
in
colonic
contents6
but
these
have
received
little
attention
in
previous
studies.
In
this
study,
we
have
investigated
the
effects
of
a
range
of
short
chain
fatty
acids
on
a
new
human
colon
carcinoma
cell
line
(LIM1215).'
It
has
pre-
viously
been
shown
by
Kim
and
his
coworkers
that
butyrate
increases
the
concentrations
of
alkaline
phosphatase
and
CEA
in
other
cell
lines
derived
from
colonic
and
rectal
carcinomas.
22-26
Butyrate
induced
various
characteristics
of
dif-
ferentiation
in
LIM
1215
cells.
The
doubling
time
of
the
cells
was
increased
markedly
and
the
cloning
efficiency
was
decreased.
Both
the
number
of
cells
producing
alkaline
phosphatase
and
the
total
amount
of
alkaline
phosphatase
being
produced
increased
markedly.
Concentrations
of
CEA
in-
creased
for
three
to
four
days
after
exposure
to
butyrate,
but
the
rise
was
not
as
sustained
as
for
alkaline
phosphatase.
Carcinoembryonic
antigen
concentrations
may
be
much
higher
in
colonic
tumours
than
in
normal
colon,
but
paradoxically,
in
tissue
culture
studies
CEA
concentrations
are
higher
in
tumour
cell
lines
which
are
well
differentiated
than
in
lines
derived
from
poorly
differentiated
or
anaplastic
tumours
as
previously
shown
by
Shi
et
al.
25
Butyrate
also
induced
dipeptidyl
peptidase
activity
in
this
cell
line
in
a
similar
manner
to
that
previously
observed
in
other
colon
carcinoma
cell
lines
by
Chung
et
al.27
Although
the
increase
in
the
total
level
of
the
enzyme
was
only
35%
which
is
modest
when
compared
with
the
60(0%
increase
in
alkaline
phosphatatse,
the
number
of
cells
with
detectable
concentrations
of
DPPIV
increased
from
3(YO-6()%
using
immunofluorescence.
The
effects
of
butyrate
on
the
levels
of
alkaline
phosphatase,
dipeptidyl
peptidase
and
CEA
indicates
that
buty-
rate
has
effects
on
the
expression
of
a
number
of
membrane
glycoproteins,
although
these
effects
vary
in
degree.
Although
the
mechanism
of
action
of
butyrate
in
inducing
these
changes
in
this
cell
line
are
unknown,
the
scanning
electron
microscopy
study
has
shown
that
the
increase
in
concentrations
of
alkaline
phosphtase
and
dipeptidyl
peptidase
is
not
simply
because
of
an
increase
in
the
number
of
microvilli
on
the
surface
of
these
cells
(Figs
6a
and
b).
We
have
previously
shown
that
cultures
of
LIM1215
cells
contain
two
morphologically
distinct
cell
types,
one
with
microvilli
on
the
surface
and
the
other
containing
mucous
inclusions
in
the
cyto-
plasm.'"
These
findings
suggest
that
LIM1215
cultures
contain
multipotent
cells
capable
of
dif-
ferentiating
along
either
the
absorptive
cell
pathway
or
the
goblet
cell
pathway.'
The
fact
that
butyrate
increased
the
number
of
alkaline
phosphatase
con-
taining
and
DPPIV
containing
cells
and
decreased
the
number
of
mucous
producing
cells
(Table
2)
suggests
that
butyrate
is
causing
the
LIM1215
cells
to
differentiate
along
the
absorptive
cell
pathway.
In
contrast
to
the
effects
of
butyrate,
the
other
short
chain
fatty
acids
tested,
acetate,
propionate,
valerate
or
isovalerate,
had
little
differentiating
effects
on
LIM1215
cells.
This
finding
is
of
interest
as
acetate
and
propionate
occur
in
the
colon
at
higher
levels
than
butyrate'
and
all
three
are
known
to
be
metabolised
by
enterocytes.7
These
findings
suggest
that
butyrate,
but
not
acetate
or
propionate,
has
a
dual
role
in
vivo
acting
as
both
an
energy
source
and
a
differentiating
agent.
This
possible
dual
role
of
butyrate
is
of
particular
interest
because
of
the
possible
prophylactic
value
of
dietary
fibre
in
decreasing
the
incidence
of
colonic
cancer.
When
fibre
is
metabolised
by
the
colonic
microflora,
the
major
metabolite
is
butyrate.6
It
might
therefore
be
postulated
that
a
major
role
of
dietary
fibre
is
to
raise
levels
of
colonic
butyrate
thus
increasing
the
differentiation
pressure
in
colonic
enterocytes.
This
theory
has
some
indirect
support
from
recent
findings
that
butyrate
concentrations
are
lower
in
the
sigmoid
and
descending
colon6
(and
M
Lawson
personal
communication)
the
region
where
the
incidence
of
colon
cancer
is
highest.
We
thank
Miss
Tina
Taranto,
Ms
Jenny
Jones,
Ms
Joy
Melny
and
Mr
Chris
Lloyd
for
their
expert
technical
assistance.
We
also
wish
to
thank
Mr
L
Wilson
for
his
assistance
with
the
scanning
electron
microscopy
studies.
We
would
also
like
to
thank
Mr
D
M
A
Francis,
Department
of
Surgery,
University
of
Melbourne,
for
providing
the
clinical
details
of
the
patient.
Part
of
this
work
was
funded
by
grants
to
Dr
G
P
Young
from
the
Anti-Cancer
Council
of
Victoria
and
the
McGauran
Trust.
References
1
Whitehead
RH,
Macrae
FA,
St
John
DJB,
Ma
J.
A
colon
cancer
cell
line
(LIM
1215)
derived
from
a
patient
with
inherited
nonpolyposis
colorectal
cancer.
J
Natl
Cancer
Inst
1985;
74:
759-65.
2
Kim
YS,
Tsao
D,
Siddiqui
B,
Whitehead
JS,
et
al.
Effects
of
sodium
butyrate
and
dimethylsulfoxide
on
biochemical
properties
of
human
colon
cancer
cells.
Cancer
1980;
45:
1183-92.
3
Leavitt
J,
Barrett
JC,
Crawford
BD,
Ts'ao
POP.
Butyric
acid
suppression
of
the
in
vitro
neoplastic
state
of
Syrian
hamster
cells.
Nature
1978;
271:
262-5.
5
Prasad
KN,
Sinha
PK.
Effect
of
sodium
butyrate
on
mammalian
cells
in
culture:
a
review.
In Vitro
1976;
12:
125-31.
6
Cummings
JH.
Short
chain
fatty
acids
in
the
human
colon.
Gut
1981;
22:
763-79.
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

Effects
of
short
chain
fitty
acids
on
a
new
human
colon
carcinoma
cell
line
(LIM1215)
1463
7
Roediger
WEW.
Utilization
of
nutrients
by
isolated
epithelial
cells
of
the
riat
colon.
Gastroenterologv
1982:
83:
424-9.
8
Whitehead
RH,
Bertoncello
1.
Webher
LM,
Pedersen
JS.
A
new
human
breast
carcinomra
cell
line
(PMC42)
with
stem
cell
characteristics.
1.
Morphologic
character-
ization.
J
Nat!
C'anicer
Inist
1983:
70:
649-61.
9
Young
GP,
Rose
IS,
Cropper
S.
Seetharam
S,
Alpers
DH.
Hepatic
clearance
of
rat
plasmai
intestinal
alkaline
phosphatase.
Am1
J
Plh,siol
1984;
247:
G419-426.
1()
Bradford
M.
A
rapid
mlnd
sensitive
method
for
the
quantitatiton
of
microgriam
quantities
of
protein
utiliz-
ing
the
principle
of
protein
-dvc
binding.
Anatil
Biochemn
1976-;
72:
248-54.
11
Nagitsu
T.
Hiiio
M,
Fuyvimrndai
H,
et
ail.
New
chro-
mogenic
substrates
f'or
x-prolv
-dipeptidyl-amino-
peptidiase.
Anatil
Biocheun
1976-,
74:
46(-76.
12
Bancroft
JD.
Enzyme
histochemistrv.
In:
Bancroft
JD.
Stevens
A,
eds.
Thleory
01t(1
pf)ractice
of
histological
techniques.
Edinburgh:
Churchill
Livingstone.
1982:
379-405.
13
Ma
J.
De
Boer
WG,
Navman
.1.
Intestinal
mucinous
subst,ances
in
gatstric
intestinal
metaplasia
aind
carcino-
ma
studied
by
immunotluorescence.
Cancer
1982;
49:
1664-7.
14
Hauri
H-P,
Sterchi
EE,
Binez
D,
Fransen
JAM,
Marxer
A.
Expression
and
intracellular
transport
of
microvillus
membrane
hydrolasis
in
human
intestinal
epithelial
cells.
J
Cell
Biol.
1985;
101:
838-51.
15
Leder
A,
Leder
P.
Butyric
acid,
at
potent
inducer
of
erythroid
differentiation
in
cultured
erythroleukemia
cells.
Cell
1975;
5:
319-22.
16
Kruh
J.
Effects
of
sodium
butyrate.
a
new
pharmacolo-
gical
algent.
on
cells
in
culture.
Mol
Cell
Biochein
1982;
42:
65-82.
17
Friend
C,
Scher
WW,
tiolland
JG,
Sato
I.
Hemoglo-
bin
synthesis
in
murine
virus-induced
leukemiia
cells
in
vitro:
stimulation
of
erVthroid
differentiation
hy
dimethylsulfoxide.
Proc
Natl
Acad
Sci
USA
1971;
68:
378-82.
18
Reuben
RC,
Rifkind
RA,
Marks
P.
Chemically
in-
duced
murine
erythroleukemic
diffcrentiiation.
Biochirn
Biophl's
Acuta
1980(;
605:
325-46.
19
Riggs
MG.
Whittaker
RG.
Neumainn
JR.
Ingram
VM.
n-Butyrate
causes
histone
modifications
in
HcLa
and
Friend
erythroleukaemia
cells.
Natitre
1977;
268:
462-4.
20
Collins
SJ,
Ruscetti
FW,
Gallagher
RE,
Gallo
RC.
Terminal
differentiation
of
human
promyelocytic
leukemia
cells
induced
by
dimethyl
sulphoxide
and
other
polar
compounds.
Proc
Natl
Acad
Sci
USA
1978;
75:
2458-62.
21
Kyritsis
A,
Joseph
G.
Chader
GJ.
Effects
of
butyriate,
rctinol
and
retinoic
acid
on
humnan
Y-79
retinoblastoma
cells
growing
in
monolaver
culture.
J
Nail
Cancer
lnst
198X);
73:
649-54.
22
Vidali
G,
Bofftat
LC.
Bradburv
EM,
Allfrey
V(G.
Butrycate
suppression
ot
histone
deacylation
leaids
to
aIccumulations
of
multilcetvlated
formns
of
histoncs
113
and
H4
and
increiased
DNaise
1
sensitivity
ot
the
associated
DNA
sequences.
Proc
Nail
Acadci
Sci
USA
1978;
75:
2'239-43.
23
Scaly
L.
Chalklev
R.
Efftect
of
sodiiutil
butyratc
on
histone
modification.
Cell
1978;
14:
115-'1.
24
Tsaio
D,
Morita
A.
Bella
A,
Luu
P.
Kim
YS.
Differentiatial
effects
of
sodium
butyrate,
dimethyl
sulfoxide
and
retinoic
acid
on
membrane-associlted
antigen,
enzytnes
and
glycoproteins
of
humain
recttl
aidenocarcinoma
cells.
Cancer
Res
1982;
42:
1(052-8.
25
Shi
ZR.
Tsiao
D.
Kim
YS.
Subcelluliar
distribution.
synthesis
and
release
of
carcinoembrvonic
antigeni
in
cultured
humain
colon
(adenocarcinomra
cell
lines.
Cancer
Re.s
1983;
43:
4045-9.
26
Tsao
D.
Shi
Z.
Wong
A.
Kim
YS.
Effect
of
sodiumn
butyrate
on
carcinoembryonlic
antigen
production
by
humatn
colonic
adcnocarcinomal
cells
in
culture.
CaIwer
Res
1983;
43:
1217-22.
27
Chung
YS.
Song
IS.
Erickson
RH.
Sleisenger
MH.
Kim
YS.
Effect
ot
growth
and
sodiumii
butyrate
on
brush
border
memnbrane-associated
hydrolases
in
human
colorectal
cancer
cell
lines.
C(ancer
Res
1985;
45:
2976-82.
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from

doi: 10.1136/gut.27.12.1457
1986 27: 1457-1463Gut
R H Whitehead, G P Young and P S Bhathal
line (LIM1215).
a new human colon carcinoma cell
Effects of short chain fatty acids on
http://gut.bmj.com/content/27/12/1457
Updated information and services can be found at:
These include:
References http://gut.bmj.com/content/27/12/1457#related-urls
Article cited in:
service
Email alerting
article.
Sign up in the box at the top right corner of the online
Receive free email alerts when new articles cite this article.
Collections
Topic
(2915 articles)Colon cancer 
collections
Articles on similar topics can be found in the following
Notes
http://group.bmj.com/group/rights-licensing/permissions
To request permissions go to:
http://journals.bmj.com/cgi/reprintform
To order reprints go to:
http://group.bmj.com/subscribe/
To subscribe to BMJ go to:
group.bmj.com on July 14, 2011 - Published by gut.bmj.comDownloaded from