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Carcinogenesis vol.24 no.3 pp.605–611, 2003
Regional response leading to tumorigenesis after sulindac in small and large intestine
of mice with Apc mutations
Kan Yang1,6, Kunhua Fan1, Naoto Kurihara1,
Hiroharu Shinozaki1, Basil Rigas2, Leonard Augenlicht3,
Levy Kopelovich4, Winfried Edelmann3,
Raju Kucherlapati5 and Martin Lipkin1
1Strang
6To
whom correspondence should be addressed
Email: [email protected]
Sulindac and other NSAIDs have been widely studied as
potential chemopreventive agents for colon cancer. Shortterm studies have shown adenomatous polyps to regress in
patients with familial adenomatous polyposis (FAP). In this
study the effect of sulindac on cancer as an endpoint was
evaluated in ApcMin mice, a preclinical model of FAP with
an Apc mutation in codon 850 that leads to gastrointestinal
adenomas and carcinomas. Three groups of mice were
studied all of which were fed AIN-76A diet: one group was
fed AIN-76A diet alone, a second group received sulindac
200 p.p.m. premixed in the diet and a third group received
sulindac 180 p.p.m. added in drinking water. ApcMin mice
were killed 9 weeks after feeding was initiated. Mice
receiving sulindac developed fewer tumors in the intestine
overall; the major decrease in tumor development after
sulindac was seen in the small intestine regardless of route
of administration. In the large intestine, however, sulindac
significantly increased the incidence, multiplicity and
volume of tumors in the colon of ApcMin mice, a regional
response to sulindac differing from previous reports.
Quantitative measurements of apoptosis, Bax and Bcl-xL
protein expression in the ApcMin mice revealed the ratio
of Bax/Bcl-xL expression and apoptosis increased in the
small intestine but decreased in the cecum, consistent with
the regional tumorigenesis observed after sulindac. These
findings thus suggest involvement of Bax and apoptosis
in tumors developing after sulindac treatment in this
mouse model.
Introduction
Several lines of evidence indicate the value of non-steroidal
anti-inflammatory drugs (NSAIDs) as potential chemopreventive agents for colorectal cancer. Long-term administration of
these compounds roughly halves the incidence of, and mortality
from colon cancer in humans (1–3). Sulindac, one of the
NSAIDs, is widely used for its anti-inflammatory properties
and has been considered a potential chemopreventive agent
for treatment of patients with familial adenomatous polyposis
Abbreviations: FAP, familial adenomatous polyposis; Apc, adenomatous
polyposis coli; ApcMin, ApcMin⫹/–; NSAIDs, non-steriodal anti-inflammatory drugs.
© All rights reserved. Oxford University Press 2003
Materials and methods
Animals and diets
Thirty ApcMin mice (Jackson Laboratories, Harbor, ME), ~4 weeks old, were
randomly divided into three groups and fed one of three diets: AIN-76A
diet alone, AIN-76A diet with 200 p.p.m. sulindac premixed, and AIN-76A
diet with 180 p.p.m. sulindac added to drinking water. The AIN-76A diet
provides 3.9 kcal/g and consists of corn oil 5% (12% of calories), calcium
0.52 mg/g (1.3 mg/kcal), phosphorus (as PO4) 0.4 mg/g (1.1 mg/kcal) and
vitamin D3 1.0 IU/g (0.26 IU/kcal). ApcMin mice were maintained on their
respective AIN-76A or treatment diets for 9 weeks. The duration of treatment
was dictated by the lifespan of 4–5 months of the mouse strain (18,19). The
average consumption of sulindac was 0.6 mg/day/mouse, an amount equivalent
to the dose that inhibits tumor growth reported in ApcMin mice (23). Mice
were weighed and feces were tested for gastrointestinal bleeding (hemoccult)
at baseline, and weekly thereafter, until animals were killed.
Evaluation of tumor development
Mice were killed by cervical dislocation, the gastrointestinal tract was removed,
opened longitudinally and the contents were washed in cold phosphatebuffered saline (PBS) pH 7.4. The mucosa was flattened on filter paper and
fixed in 10% neutral-buffered formalin. Each specimen was examined under
a dissecting microscope. Tumors were numbered and their location was
recorded. Tumor volume, measured using a grid in the eyepiece of the
microscope, was expressed in units of mm3. The flat mucosa of the stomach,
duodenum, cecum, proximal and distal colon was processed for further study.
Representative tumors from the small intestine and all the tumors in the
cecum and colon were sampled and processed. All other internal organs and
lymph nodes were examined for tumors. Sections of paraffin-embedded tumor
605
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Cancer Research Laboratory at The Rockefeller University,
1230 York Avenue, New York, NY 10021, USA, 2American Health
Foundation, Valhalla, NY 10595, USA, 3Albert Einstein College of
Medicine, Bronx, NY 10461, USA, 4Division of Cancer Prevention,
National Cancer Institute, Bethesda, MD 20892, USA and 5Division of
Genetics, Brigham and Women Hospital, Boston, MA 02115, USA
(FAP) (4–6). In addition to inhibiting aberrant crypt foci in
the colon, a precursor of adenoma and cancer (7), sulindac
has decreased the number and size of adenomas in humans
with FAP (8–15) without having complete regression in all
studies (10–12,14,15). In rodents sulindac inhibits colonic
carcinogenesis induced by chemical carcinogens (16,17). The
establishment of mouse models with targeted Apc gene
mutations makes it possible to carry out chemopreventive
studies on models of human FAP (18–22). The effects of
NSAIDs including sulindac on intestinal tumor development
in these mouse models are similar to those of humans,
with decreased tumor number and size and without complete
disappearance (23–29). Some attention has been given to
colorectal cancer developing in patients with FAP after they
received sulindac treatment (13,30–33).
In view of the emerging role of sulindac and similar
pharmaceutical agents as potentially important chemopreventive agents for colon cancer, we carried out a detailed study
of the effects of sulindac on tumorigenesis in ApcMin mice,
an animal model of human FAP. In this report we provide
evidence that, while sulindac did indeed decrease the number
and volume of tumors in the small intestine, it simultaneously
increased tumor incidence, number and volume in the large
intestine.
To explore mechanisms that might contribute to this regional
response to sulindac, we carried out measurements of apoptosis,
and Bax and Bcl-xL in the small intestine, cecum and distal
colon. Regional responses of apoptosis, and Bax/Bcl-xL expression after sulindac treatment were demonstrated in the
ApcMin mice.
K.Yang et al.
Table I. Incidence, number and volume of intestinal tumors
Group
n
Overall
Small intestine
Large intestine
A. Incidence
AIN-76A
Sulindac in AIN-76A
Sulindac in drinking water, mice fed AIN-76A
10
10
10
10 (100%)
10 (100%)
10 (100%)
10 (100%)
7 (70%)
10 (100%)
3 (30%)
10 (100%)a
10 (100%)a
B. Tumor number per mouse, mean ⫾ SEM
AIN-76A
Sulindac in AIN-76A
Sulindac in drinking water, mice fed AIN-76A
10
10
10
37.1 ⫾ 5.3
5.5 ⫾ 2.4b
14.9 ⫾ 3.9a
36.7 ⫾ 5.3
3.4 ⫾ 2.2b
11.1 ⫾ 3.7a
0.4 ⫾ 0.2
2.1 ⫾ 0.4a
3.8 ⫾ 0.4a
C. Tumor volume (mm3), mean ⫾ SEM (median)
AIN-76A
Sulindac in AIN-76A
Sulindac in drinking water, mice fed AIN-76A
10
10
10
43.2 ⫾ 5.1 (42.3)
3.6 ⫾ 0.6 (3.5)b
6.0 ⫾ 1.4 (4.5)b
41.9 ⫾ 5.1 (41.6)
0.6 ⫾ 0.3 (0.02)a
1.7 ⫾ 1.0 (0.6)a
1.3 ⫾ 0.9 (0.0)c
3.0 ⫾ 0.5 (3.0)a,d
4.3 ⫾ 0.7 (3.9)a,d
the proteins were made in tumors and corresponding flat mucosa in those
regions of the ApcMin mice.
Apoptosis
Apoptosis originated as a morphological phenomenon, and measurements in
the present study were based on morphologic features. Characteristic features
required for identification of apoptotic bodies included: a single, condensed
small cell with or without cytoplasm (apoptotic body) or a cluster of apoptotic
bodies inside another healthy cell (host cell), with or without a halo around
the apoptotic body. Measurements were carried out on well-oriented intestinal
crypts of flat mucosa. Cells on the mucosal surface of the proximal and distal
colon were also included. Twenty-five crypts (50 crypt columns) were scored
for each mouse. The apoptotic cells per crypt column were computed.
Apoptotic cells in the villi of small intestine were measured separately. The
number of apoptotic cells in each villous column was scored. Twenty five
villi (50 villous columns) were studied for each mouse. The number of villous
columns with apoptotic cells and number of apoptotic cells per villous column
were calculated. In tumors, 1000 tumor cells or more were scored for
apoptotic bodies.
Fig. 1. Tumorigenesis in the gastrointestinal tract of ApcMin mice after
sulindac. (A) tumor incidence, (B) tumor multiplicity, (C) tumor volume
(*P ⬍ 0.01; **P ⬍ 0.001, sulindac group compared with controls).
tissues and 15 serial sections from flat mucosa of each location sampled were
examined histologically after staining with hematoxylin and eosin. Diagnoses
were based on the WHO classification system (34).
To determine the role of apoptosis and related proteins Bax/Bcl-xL in tumor
development in both small and large intestine, measurements of apoptosis and
606
Evaluation of Bax and Bcl-xL expression
Expression of Bax and Bcl-xL was evaluated on immunohistochemical preparations of tumors taken from small and large intestine and flat mucosa of
duodenum, cecum and distal colon of ApcMin mice in sulindac and control
groups. The antibodies to Bax (N-20) and Bcl-xL (A-20) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Six slides with five serial tissue
sections per slide were cut from a paraffin block and labeled. To avoid
variation of staining intensity, all tissue-slides were stained at the same time,
and all tissue sections were cut by one technologist with the same microtome.
Three slides with an odd number (nos 1, 3 and 5) were used for immunohistochemical staining for Bax and Bcl-xL and the first two slides with an even
number (nos 2 and 4) were stained for H&E for apoptosis study. On the slides
with odd numbers the specific antibody to Bax and Bcl-XL was applied to
every other section in each slide. This parallel staining for two different
antibodies facilitated the data being more comparable. The last slide (no. 6)
was used for negative control on which neither antibody to Bax nor to BclXL was added to tissue sections as immunohistochemical staining. Measurements were carried out with a computer-assisted cell image analyzer (Samba
4000). This system uses a color CCD camera to separate red (R), green (G)
and blue (B) colors and convert these colors to digital form. The size of the
resulting pixels was 0.25 µm under a magnification of 40⫻. By using a
software program, immuno 4.04 per Area Program for cytoplasmic labeling
measurement, it was possible to assay the size of positive areas (Labeling
Index) and staining intensity (Mean Optical Density) of a given area in an
immunohistochemical preparation. Through segregation the assay was confined
only to epithelial cells, i.e. in a crypt column of flat mucosa, and to neoplastic
glands of a tumor. Ten crypts (20 crypt columns), well oriented, were measured
from each intestinal segment of flat mucosa for each mouse. For tumors, 1000
cells from different areas were randomly selected for measurement. Bax and
Bcl-XL protein expression was assayed separately in two differently staining
tissue sections nearby on one slide for each mouse or tumor. The data were
automatically generated with an arbitrary unit (a.u.) including percentage of
positive area, staining intensity and overall expression of a combination of
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n, number of mice studied.
Comparisons are made to corresponding control (AIN-76A diet, no treatment) using two-tailed Fisher exact probabilities test for tumor incidence; Mann–
Whitney and exact binomial calculation for tumor number and volume: aP ⬍ 0.01; bP ⬍ 0.001. Comparisons between tumor volume in the large intestine and
small intestine: cP ⬍ 0.001; dP ⬍ 0.01.
Sulindac induces colonic tumors
Table II. Distribution of tumors found in the large intestine
Group
Mean number of tumors per mouse
Overall
AIN-76A
Sulindac in AIN-76A
Sulindac in drinking water, mice fed AIN-76A
0.4 ⫾ 0.2
2.1 ⫾ 0.4a
3.8 ⫾ 0.4a
Right side
Left side
Subtotal
Cecum
PC
DC
0.3 ⫾ 0.2
1.8 ⫾ 0.3a,b
3.6 ⫾ 0.5a,b
0.2 ⫾ 0.1
1.3 ⫾ 0.2a
3.3 ⫾ 0.4a
0.1 ⫾ 0.1
0.5 ⫾ 0.3
0.3 ⫾ 0.3
0.1 ⫾ 0.1
0.3 ⫾ 0.2
0.2 ⫾ 0.1
aBy Mann-Whitney test or Exact Binomial Calculation: compared with AIN-76A
bCompared with the left side of colon of the same diet group: P ⬍ 0.01.
for each mouse strain: P ⬍ 0.01.
positive area and staining intensity (percentage of positive area⫻staining
intensity). The overall expression was used for the comparisons. The ratio of
overall expression of Bax and Bcl-xL are reported for each group, i.e. ratio of
Bax and Bcl-XL expression ⫽ mean overall expression of Bax/mean overall
expression of Bcl-XL. Comparisons of protein expression between treatment
and control groups were based on the ratio.
intestine tumor volume decreased 99 and 96% in two sulindac
groups, respectively, over the controls with P ⬍ 0.01 for both
(0.6 and 1.7 versus 41.9 mm3). However, in the large intestine,
tumor volume increased 1.3- and 2.3-fold in the sulindactreated groups, P ⬍ 0.01 for both compared with controls (3.0
and 4.3 versus 1.3 mm3). In both sulindac-treated groups,
tumor volume was greater in the large intestine than in the
small intestine with P ⬍ 0.01 for both, but it was opposite in
the control group (Table I and Figure 1C).
Distribution and histology of the large intestinal tumors. The
large intestinal tumors that were unexpectedly found in the
sulindac-treated groups were studied in detail, with respect to
their location and their histological features.
Location of tumors in the large intestine. The vast majority of
large intestinal tumors were located in the right side of the
large intestine, especially in the cecum (Table II and Figure
2A). The mean number of large intestinal tumors in the control
group was low (0.4). The preponderance of tumors in the right
colon increased in the sulindac-treated groups. The number of
tumors in the right side increased 5- and 11-fold in both
sulindac-treated groups, P ⬍ 0.01 for both compared with
controls (1.8 and 3.6 versus 0.3). The tumor number in the
right side was significantly higher than in the left side in both
sulindac groups, P ⬍ 0.01, but not in the controls (Table II).
Thus, the markedly increased tumor incidence in the large
intestine was due to tumors developing in the right colon,
especially in the cecum.
Histological types of tumors in the large intestine. All 60
tumors found in the large intestine from all three groups were
evaluated histologically (Table III). Control ApcMin mice had
four tumors found in the colon with one invasive carcinoma
and three benign tubular adenomas. After sulindac treatment,
invasive carcinomas increased ~8- to 10-fold in both groups.
Invasion of the submucosa was found in all 20 adenocarcinomas
in sulindac-treated groups (Figure 2B). The potentially malignant villous adenoma increased in the proximal colon in one
sulindac group, and flat adenomas were greatly increased and
mostly seen in the cecum of mice treated with sulindac.
These findings demonstrated a complex effect of sulindac
on tumorigenesis in this preclinical mouse model: tumors
inhibited in the small intestine and tumors increased in the
large intestine. Similar effects of sulindac on tumorigenesis
were also observed in Apc1638N mice (26), another mouse
model of human FAP; the sulindac administration and diets
fed were the same as for ApcMin mice, and the feeding
duration was 6 months longer than for ApcMin mice because
the lifespan is 12–15 months in Apc1638N and 4–5 months in
ApcMin mice (26).
Mean ⫾ SEM. PC, proximal (upper half) colon; DC distal (lower half) colon.
Results
All tumors found in the ApcMin mice were in the intestinal
tract. There were no tumors in the stomach and none were
detected in the lungs, liver, kidneys or lymph nodes of
these mice.
Tumorigenesis
Tumor incidence. We determined the tumor incidence in the
small and large intestine as well as the overall tumor incidence,
i.e. tumor incidence in a group of mice regardless of anatomical
location of the tumor in the intestinal tract (Table I and Figure
1A). In the small intestine the tumor incidence in the group
given sulindac premixed in the solid diet decreased 30%
compared with the control group (P ⬎ 0.05). However, in the
large intestine, the tumor incidence was 2.3-fold increased in
both sulindac-treated groups (100 versus 30% with P ⬍ 0.01)
compared with controls. The overall tumor incidence was
100% in each of the three groups having tumors (Table I and
Figure 1A).
Number of intestinal tumors per mouse (multiplicity). The
overall number of tumors per mouse decreased 85 and 60%
in both sulindac-treated groups compared with controls with
P values ⬍0.001 and ⬍0.01, respectively (5.5 and 14.9 versus
37.1) (Table I and Figure 1B). In the small intestine the number
of tumors per mouse decreased 90 and 70% in both sulindac
groups compared with controls with P values 0.001 and 0.01,
respectively (3.4 and 11.1 versus 36.7, representing mean
tumor number in mice fed sulindac in solid food and in
drinking water versus tumors in control group (same for below
if not specified). In the large intestine, however, changes in
the tumor number were in the opposite direction from those
seen in the small intestine. The tumor number increased
4.3- and 8.5-fold in the two sulindac-treated groups with both
P ⬍ 0.01 compared with controls (2.1 and 3.8 versus 0.4).
Tumor volume. The overall tumor volume decreased after
sulindac treatment in ApcMin mice. The mean overall volume
decreased 92 and 86% in the two sulindac groups compared
with controls with both P ⬍ 0.001 (3.6 and 6.0 versus 43.2
mm3). Similar to the overall tumor volume, in the small
607
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Statistical tests
Comparisons between the study and control groups were carried out using
Fisher exact probabilities, Mann–Whitney test, and binomial calculations.
Significant difference was considered as P ⬍ 0.05.
K.Yang et al.
608
Discussion
This study demonstrated that the chemopreventive agent sulindac, when administered to ApcMin mice, caused decreased
number and size of tumors in the small intestine, consistent
with reports in the literature (23–25,27,28). The reduction of
tumors in the small intestine provided an important internal
control to findings observed in the large intestine. An overall
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Fig. 2. Intestinal tumors in sulindac-treated ApcMin mouse. (A) Two gross
tumors elevated above the cecal mucosa (arrows), with ulceration in the
center (1.75⫻ original magnification). (B) Histological features of an
adenocarcinoma of cecum from the same mouse as (A), with irregular
neoplastic glands infiltrating in the submucosa of intestinal wall (arrows)
with ulceration on the surface. Hematoxylin and eosin, 200⫻ original
magnification. (C) Immunohistochemical preparation showing strong Bax
expression in an adenoma in the small intestine of a mouse after sulindac
treatment, 100⫻ original magnification.
Apoptosis
Flat mucosa. To evaluate apoptosis during tumor development,
apoptotic bodies were counted with criteria noted in the
Materials and methods. Findings are presented in Table IV.
Apoptosis was counted in the three groups of mice in intestinal
crypt epithelial cells of flat mucosa of duodenum, cecum and
distal colon. The number of apoptotic cells per crypt column
decreased 44% in the cecum (0.34 versus 0.61, P ⬍ 0.001)
and 33% in the distal colon (0.14 versus 0.21, P ⬍ 0.01), but
did not significantly change in duodenal crypts (0.08 versus
0.10, P ⬎ 0.05) of mice after sulindac treatment. However, in
villi of duodenum the number of villous columns with apoptotic
cells increased 30% (19.0 versus 14.6, P ⬍ 0.05) and the
number of apoptotic cells per villous column increased 40%
(0.21 versus 0.15, P ⬍ 0.01) in mice after sulindac. The data
thus indicated that in response to sulindac treatment apoptosis
differed in flat mucosa of the small and large intestine in
ApcMin mice.
Tumors. Apoptosis was measured in tumors located in small
and large intestine of sulindac-treated and control groups of
mice. After sulindac treatment the percentage of apoptotic
cells increased 1.4-fold in small intestinal tumors (0.0156
versus 0.0065, P ⬍ 0.001), and decreased 34% in the cecal
tumors (0.0100 versus 0.0151, P ⬎ 0.078). The reduction of
apoptosis in cecal tumors did not reach statistical significance,
which might be due to only two tumors in the control group.
Thus, in the cecum of ApcMin mice apoptosis decreased in
flat mucosal cells with a trend to decrease in tumor cells, and
increased in small intestinal villous epithelial cells and tumor
cells during tumorigenesis.
Expression of Bax and Bcl-xL
Flat mucosa. Quantitative measurements by computer-assisted
cell image analysis showed Bax expression increased and BclXL decreased in epithelial cells in the duodenum with the
opposite result in the cecum after sulindac treatment. The
overall expression of proteins related to apoptosis in epithelial
cells, presented as a ratio of Bax to Bcl-xL increased in the
small intestine and decreased in the cecum after sulindac. The
ratio increased 1.2-fold in the duodenum (1.38 versus 0.62,
P ⬍ 0.05) and decreased 55% in the cecum (0.47 versus 1.04,
P ⬍ 0.001) after sulindac treatment (Figure 3A). However, no
significant difference was seen in the distal colon (0.72 versus
0.59, P ⬎ 0.05) (Figure 3A).
Tumors. Similar to the findings of Bax and Bcl-XL expression
in flat mucosa of duodenum, Bax expression increased and
Bcl-XL decreased in tumors of the small intestine, and the
opposite result was observed in the cecal tumors. Figure 2C
is representative of the small intestinal tumors showing Bax
with strong expression in neoplastic adenoma cells after
sulindac treatment. The ratio of Bax to Bcl-xL in tumors
increased 3-fold in the small intestine (1.11 versus 0.28,
P ⬍ 0.01) and decreased 79% in the cecum (0.87 versus 4.18,
P ⬍ 0.05) after sulindac treatment in ApcMin mice (Figure 3B).
Sulindac induces colonic tumors
Table III. Histologic types of large intestinal tumors
Group
Number of tumors found in the large intestine
AIN-76A
Sulindac in AIN-76A
Sulindac in drinking water mice fed AIN-76A
Total
Adenocarcinoma
Villous adenoma
Flat tubular adenoma Tubular adenoma Micro-adenoma
4
21
35
1
9
11
0
4
0
0
4
22
3
3
1
0
1
1
Table IV. Apoptosis of intestinal epithelial cells in flat mucosa and tumors
of ApcMin mice after receiving control AIN-76A diet and AIN-76A with
sulindac
Control AIN-76A
AIN-76A with sulindac
0.08 ⫾ 0.01
0.34 ⫾ 0.01a
0.14 ⫾ 0.01b
Percent of villous columns with apoptotic cells
Duodenum
14.6 ⫾ 1.1
19.0 ⫾ 1.4c
Number of apoptotic cells/villous column
Duodenum
0.15 ⫾ 0.01
0.21 ⫾ 0.02b
Tumors
Percent of apoptotic cells in 1000 tumor cells
Small intestine
0.65 ⫾ 0.08
Cecum
1.51 ⫾ 0.79
1.56 ⫾ 0.22a
1.00 ⫾ 0.08d
By Mann–Whitney test: compared with corresponding organ with AIN-76A:
aP ⬍ 0.001; bP ⬍ 0.01; cP ⬍ 0.05.
Compared with tumors of small intestine after sulindac; dP ⫽ 0.078.
Mean ⫾ SEM. The number of tumors studied was 17 in control group
including 15 in the small intestine and two in the cecum; 27 in the sulindac
group including 12 in the small intestine and 15 in the cecum.
protective effect of sulindac against tumor development in the
intestine as a whole was observed. When sulindac was premixed in AIN-76A diet or supplied in drinking water it
decreased the overall tumor number (85 and 60%), and
decreased tumor volume (92 and 86%) in the intestine of the
ApcMin mice, findings consistent with previous literature.
However, unexpectedly sulindac induced tumor formation
in the colon, regardless of the route of administration: premixed
in a solid control AIN-76A diet, or added to the drinking
water. Findings thus showed a complex effect of sulindac
in the intestinal tract of mice with this Apc mutation, a
chemopreventive effect in the small intestine, but a tumorenhancing effect in the right side of the large intestine.
The increase of tumors in the large intestine of ApcMin
mice after sulindac was profound, indicated by increased tumor
incidence, multiplicity, volume and higher grade of malignancy
in the colon, differing from sulindac effects in small intestine.
In humans, carcinomas are known to develop in FAP patients
maintained on sulindac treatment (13,30–33), appearing endoscopically as flat, ulcerated lesions. By coincidence these
findings in ApcMin mice after sulindac are similar; the occurrence of flat adenomas in these mice is of particular interest
as they have been found to occur with increased malignant
potential in humans with a hereditary predisposition to colon
cancer (35–38).
To evaluate the comparative tumor response after sulindac
treatment in colon and small intestine, the tumor development
ratio of treatment group/control group was analyzed. The
Fig. 3. Overall expression of proteins Bax and Bcl-xL presented as ratio of
Bax/Bcl-xL in duodenum, cecum and distal colon. (A) In flat mucosa. (B) In
tumors of small intestine and cecum. (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍
0.001, sulindac group compared with controls.)
relative benefit/harm (RB/H) was calculated as: parameter
studied in small intestine⫻large intestine in the treatment
group/parameter studied in small intestine⫻large intestine in
the control group. Treatment was of benefit with RB/H ⬍1,
and was harmful with RB/H ⬎1. A 95% confidence interval
(95% CI) was also calculated using log transformed means to
further evaluate the significance of RB/H. A beneficial effect
was suggested in tumor multiplicity after treatment (RB/H ⫽
0.49) but without statistical significance (95% CI ⫽ 0.091–
2.583). However, a beneficial effect was observed with a
reduction of tumor volume after treatment in the group
receiving sulindac in AIN-76A (RB/H ⫽ 0.03, 95% CI ⫽
0.006–0.185). Mice receiving sulindac supplied in drinking
water also had a beneficial effect with the tumor volume
decreasing, but not tumor number (data not shown). Findings
thus indicated a beneficial relative tumor response in colon
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Flat mucosa
Number of apoptotic cells/crypt column
Duodenum
0.10 ⫾ 0.02
Cecum
0.61 ⫾ 0.04
Distal colon
0.21 ⫾ 0.02
K.Yang et al.
Acknowledgements
The work was supported by NIH Awards CN65031, R01-CA87559, U01-CA84301 and the Ann E.Woodward Foundation.
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