Cancer Therapy: Preclinical
The Intestinotrophic Peptide, GLP-2, Counteracts Intestinal
Atrophy in Mice Induced by the Epidermal Growth Factor
Receptor Inhibitor, Gefitinib
Kristine Juul Hare,1 Bolette Hartmann,2 Hannelouise Kissow,1 Jens Juul Holst,2 and Steen Seier Poulsen1
Abstract
Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been
introduced as antitumor agents in the treatment of cancers overexpressing the receptor. The
treatment has gastrointestinal side effects which may decrease patient compliance and limit the
efficacy. Glucagon-like peptide-2 (GLP-2) is an intestinal hormone with potent intestinotrophic
properties and therapeutic potential in disorders with compromised intestinal capacity. The
growth stimulation is highly specific to the gastrointestinal tract, and no effects are observed
elsewhere. The aim of this study was to examine whether the inhibition of the EGFR induces
intestinal atrophy and if this can be counteracted by treatment with GLP-2.
Experimental Design: Mice were treated for 10 days with either gefitinib orally, GLP-2 as
injections, or a combination of both. After sacrifice, the weight and length of the segments
of the gastrointestinal tract were determined, and histologic sections were analyzed by morphometric methods.
Results: A significant atrophy of the small-intestinal wall was observed after treatment with
gefitinib because both intestinal weight and morphometrically estimated villus height and crosssectional area were decreased. The same parameters were increased by GLP-2 treatment alone,
and when GLP-2 was combined with the gefitinib treatment, the parameters remained
unchanged.
Conclusions: Treatment with an EGFR tyrosine kinase inhibitor in mice results in small-intestinal
growth inhibition that can be completely prevented by simultaneous treatment with GLP-2. This
suggests that the gastrointestinal side effects elicited by treatment with EGFR tyrosine kinase
inhibitors can be circumvented by GLP-2 treatment.
The epidermal growth factor receptor (EGFR) is expressed by
approximately one-third of all human epithelial cancers,
including non – small cell lung cancer (NSCLC), prostate, breast,
colorectal, head and neck, ovarian, gastric, and pancreatic
cancers (1 – 4). The EGFR pathway contributes to a number
of processes involved in tumor survival and growth, such as
cell proliferation, inhibition of apoptosis, angiogenesis, and
metastasis, thus making it an attractive target for anticancer
therapies (5, 6).
EGFR tyrosine kinase inhibitors (EGFR-TKI) that block the
signal transduction pathways implicated in the proliferation
and survival of cancer cells and other host-dependent processes
promoting cancer growth have recently been introduced in
the treatment of cancers, especially as second- or third-line
therapy or in combination with chemotherapy. Orally active
Authors’Affiliations: Departments of 1Anatomy and 2Physiology, Panum Institute,
University of Copenhagen, Copenhagen, Denmark
Received 3/8/07; revised 5/25/07; accepted 6/4/07.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Steen Seier Poulsen, Anatomy Department B, Panum
Institute, University of Copenhagen, 3 Blegdamsvej, DK-2200 Copenhagen N,
Denmark. Phone: 45-35327253; E-mail: s.s.poulsen@ mai.ku.dk.
F 2007 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-07-0574
Clin Cancer Res 2007;13(17) September 1, 2007
EGFR-TKIs, gefitinib (Iressa) and erlotinib (Tarceva), with high
selectivity due to minimal effect on other tyrosine or serine/
threonine kinases, are under clinical development (7, 8).
Primarily, they have been tested in NSCLC, but also in
metastatic colorectal cancers, recurrent head and neck cancers,
and gliomas (for a review, see ref. 9). The potential role of the
EGFR-TKIs has not been fully determined, but large phase II
studies where gefitinib or erlotinib were given as second-line
therapy to NSCLC patients showed antitumor activity as
determined by tumor shrinkage, stabilization of disease, and
relief of symptoms, especially following erlotinib. Both EGFRTKIs are generally well tolerated: the most frequent adverse
effects noted were skin rashes and gastrointestinal symptoms
such as nausea, vomiting, and especially diarrhea. A combination of EGFR-TKI (erlotinib) and a standard chemotherapy
regimen (FOLFORI) had to be terminated due to excessive
toxicity, including grade 3 diarrhea and vomiting (10). The
occurrence of gastrointestinal symptoms is in accordance with
the supposed role of the EGF system in the regulation of growth
and differentiation in the gastrointestinal tract (11 – 13),
although a direct effect on the gastrointestinal tract by the
inhibition of the EGF system by the EGFR-TKIs has never been
shown.
Gastrointestinal side effects were observed in f80% of
patients following treatment with gefitinib, and this required
dose reduction and caused treatment delay in some patients
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GLP-2 and Gefinitib-Induced Intestinal Atrophy
(14 – 17). Both the occurrence and severity of diarrhea and skin
rashes were found to be dose dependent (15, 16, 18). Because
treatment with EGFR-TKIs most probably is going to be
prolonged—several cycles of 4-week treatment periods—it is
desirable to avoid the gastrointestinal side effects which can
influence the general well-being of the patients.
Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide
derived from the posttranslational processing of proglucagon.
It has highly specific intestinotrophic effects (19 – 22), and
following treatment with exogenous peptide, a growth response
is seen only in the intestinal system. GLP-2 is supposed to play
an important role in the regulation of the size and absorptive
capacity of the gut. GLP-2 treatment increases small bowel
weight and mucosal thickness in mice (20) by inducing a lower
rate of apoptosis in the enterocytes and a higher rate of cryptcell proliferation (19). The growth factor system activated by
GLP-2 stimulation has not yet been unambiguously identified,
although it has been shown that insulin-like growth factor I
(IGF-I; ref. 23) as well as the keratinocyte growth factor (24)
seem to be involved.
We hypothesized that unless GLP-2 exerts its growthpromoting action via the EGF system, the inhibition of
intestinal growth by EGFR-TKIs might be counteracted by the
treatment with GLP-2. In the present study, we describe the
direct effect of short-term gefitinib treatment in mice on
morphometric parameters in the various segments of the
gastrointestinal tract, and we investigate whether the possible
growth-inhibiting effects of gefitinib might be counteracted
by the simultaneous administration of GLP-2.
Materials and Methods
Animals. The animal studies were approved by the Danish Ministry
of Justice, Animal Experiments Inspectorate. Female C57bl mice (M&B)
weighing f21 g were housed in plastic-bottomed wire-lidded cages.
They were maintained throughout the course of the experiment on
water and chow (no. 1314, Altromin) ad libitum in animal facilities
with temperature (21jC) – and humidity (55%) – controlled rooms
with a light-dark cycle of 12 h each. All animals were acclimatized for at
least 1 week before the study started.
Experimetal design. Human recombinant GLP-2 (a generous gift
from L. Thim, Novo Nordisk A/S, Bagsværd, Denmark) was dissolved in
PBS containing 3.5 mg/mL Hemaccel (Behringwerke AG), which was
also used for control injections. The injection volume was 100 AL
containing 25 Ag GLP-2, given as s.c. injections twice daily every 12 h for
10 days. EGFR inhibitor, gefitinib (Iressa), kindly donated from
AstraZeneca, was prepared as a suspension (2.5 mg/mL) in 1% aqueous
Tween 80 by homogenization with glass beads for f18 h. The dose
volume was 0.2 mL administered by oral gavages twice daily every 12 h
for 10 days. The suspension was kept at room temperature, with stirring
during the experiment.
Animals were weighed and randomly allocated to the following
groups of 10: (a) PBS, (b) GLP-2 (25 Ag), (c) gefitinib (0.5 mg), and (d)
GLP-2 (25 Ag) + gefitinib (0.5 mg). Animals in groups a and b were, in
addition, p.o. dosed with 0.2 mL water containing 1% Tween 80.
Animals in group c had control injections with PBS. All animals were
sacrificed after 10 days of treatment. After removal of mesenteric fat and
luminal contents of the stomach and gut, the weight and length of the
small and large intestines and the weight of the stomach were recorded.
When measuring length, all intestinal segments were vertically
suspended with a 1.5-g weight to provide uniform tension.
Histologic sections and morphometric analysis. Tissue samples from
the small intestine (proximal, middle, and distal) and colon were fixed
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by immersion in ice-cold, freshly prepared buffered 4% paraformaldehyde. The fixed tissue samples were then dehydrated, embedded in
paraffin, and cut perpendicularly to the axis of their length into 10-Am
sections. The sections were stained with PAS-HE and were examined
using a Leitz Ortoplan microscope fitted with a cooled camera,
Evolution MP (MediaCybernetics). The cross-sectional area of the
mucosa and muscular layers in the small and large intestines as well as
the PAS-positive (mucus-containing) area in the stomach (indicated
as square micrometers per millimeter mucosa) and colon (indicated as
PAS-positive area in percent of the cross-sectional area) was measured.
The thickness of the stomach mucosa and height of the gastric pits,
the villus height, and crypt depth were also measured using Image-Pro
Plus 5.0. The examination and the computer analysis of the histologic
sections were done without the knowledge of the origin of tissue
samples.
Statistical analyses. The results are shown as mean F SE. Statistical
significance of the differences obtained between initial and final body
weight for each of the four treatment groups were assessed by an
unpaired t test. Comparison between groups was done by one-way
ANOVA, followed by Tukey’s post hoc analysis. Probability values of
P < 0.05 were considered significant.
Results
Body weight changes during the study. Initial and final body
weight for the four groups are summarized in Table 1. Mice
from group a (PBS) and group c (gefitinib) had a statistically
significant (P = 0.048 and P = 0.00016) lower final body
weight compared with their initial body weight (t test). PBStreated mice lost in average 1.1 g (from 20.8 to 19.7 g), and
gefitinib-treated animals lost 1.2 g (from 20.9 to 19.7 g).
GLP-2 – and GLP-2 + gefitinib – treated mice had minor and
insignificant weight losses. Statistical analysis comparing the
four treatment groups (ANOVA) showed a significant difference
(P < 0.05) in final body weight between animals in group b
(GLP-2) and group c (gefitinib).
Effects of GLP-2 and gefitinib on the weight and length of
gastrointestinal organs. There was no significant difference
between the four treatment groups in the weight of the stomach
and the length of the small intestine and the length of the colon
(data not shown). Significant differences between the groups
were found in the weight of the small intestine and the colon
(Fig. 1). The weight of the small intestine (expressed in percent
of the body weight) was significantly (P < 0.001) reduced by
17% in gefitinib-treated animals (3.58 F 0.09%) compared
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Table 1. Body weight (BW) of mice treated 10 d
with PBS, GLP-2, gefitinib, or gefitinib + GLP-2
PBS
GLP-2
Gefitinib
Gefitinib
+ GLP-2
BW initial (g) 20.8 F 0.3* 21.2 F 0.2 20.9 F 0.2c 21.0 F 0.4
BW final (g) 19.7 F 0.4b 21.0 F 0.2 19.7 F 0.2b 20.4 F 0.4
NOTE: Results are mean F SE.
*P < 0.05 compared with final body weight of PBS-treated mice
(t test).
cP < 0.0002 compared with final body weight of gefitinib-treated
mice (t test).
bP < 0.05 compared with final body weight of GLP-2 – treated mice
(ANOVA).
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Cancer Therapy: Preclinical
Fig. 1. Small-intestinal weight (A) and colonic weight (B) expressed as percent
of the body weight in PBS-, GLP-2 ^ , gefitinib ^ , and gefitinib + GLP-2 ^ treated
mice. Results are mean + SE. a, P < 0.05; A, P < 0.01; and AA, P < 0.001, compared
with gefitinib-treated mice. BB, P < 0.001, compared with GLP-2 ^ treated mice.
C, P < 0.01, compared with gefitinib + GLP-2 ^ treated mice.
with controls (4.30 F 0.06%). In contrast, the small intestine in
the GLP-2 – treated mice showed a marked increase (P < 0.001)
in small-intestinal weight (5.74 F 0.10% of body weight)
compared with controls. Animals treated with GLP-2 in
combination with gefitinib (group d) showed a significantly
increased small-intestinal weight (4.82 F 0.11%) compared
with both gefitinib-treated mice (P < 0.001) and controls
(P < 0.01). This increase was, however, significantly lower
compared with the weight increase induced by GLP-2 alone
(P < 0.001; Fig. 1A). The colonic weight was also influenced by
gefitinib treatment because the weight of the colon (in percent
of the body weight) was significantly (P < 0.001) decreased by
14% in the gefitinib-treated animals (1.10 F 0.02%) compared
with controls (1.28 F 0.05%). Also, when compared with
GLP-2 – treated animals (1.24 F 0.02%) and gefitinib + GLP-2 –
treated animals (1.23 F 0.01), the colon weight of the gefitinibtreated mice was significantly decreased (P < 0.01 and P < 0.05,
respectively). The colon weight in the GLP-2 – treated and the
GLP-2 + gefitinib – treated animals was comparable to the
controls.
Morphometric analysis. Only minor changes of the morphometric parameters of the stomach were found (Table 2). There
were no differences in the height of the mucosa between the
four groups. Gefitinib-treated animals showed a minor decrease
(not statistically significant) in the height of the gastric pits
compared with controls, whereas the amount of mucin in the
Clin Cancer Res 2007;13(17) September 1, 2007
mucosa was similar to that of the controls. Animals treated with
GLP-2 and GLP-2 + gefitinib showed increases (not statistically
significant) in both pit height and amount of mucin compared
with animals treated with gefitinib alone and control animals.
The GLP-2 + gefitinib – treated group was comparable to the
GLP-2 group.
In the small intestine, the effects of gefitinib and GLP-2
were mainly seen in the proximal part (Table 2). The crosssectional area of the proximal part of the small intestine in
gefitinib-treated mice (3.40 mm2) was similar to that of
the controls (3.27 mm2), whereas the GLP-2 – treated mice
(4.63 mm2) had a significantly increased area compared with
controls (P < 0.01) and compared with gefitinib (P < 0.01).
The proximal cross-sectional area in the GLP-2 + gefitinib –
treated (4.00 mm2) animals was increased compared with
gefitinib-treated mice and compared with controls (not
significant).
The cross-sectional area of the middle part of the small
intestine was increased in GLP-2 – treated animals both
compared with the controls and the gefitinib-treated groups
(P < 0.05; Table 2). No statistically significant differences
between the four treatment groups were detected regarding the
cross-sectional areas of the distal part of the small intestine
(Table 2).
The villus height (and thereby the small-intestinal surface
area) was the parameter exhibiting the most pronounced
changes following the treatments, and gefitinib and GLP-2
clearly had opposite effects (Fig. 2). Again, changes were most
pronounced in the proximal part of the small intestine (Fig. 3),
where the villus height was significantly decreased by 28%
(P < 0.001) following treatment with gefitinib (0.51 mm)
compared with controls (0.71 mm). In contrast, villus height
was significantly increased (P < 0.05) in GLP-2 – treated
animals (0.84 mm) compared with controls and compared
with the gefitinib-treated animals (P < 0.001). When GLP-2 was
given in combination with gefitinib, the gefitinib-induced villus
atrophy was completely counteracted. Villus height in GLP-2 +
gefitinib – treated mice (0.75 mm) was comparable to controls
but was significantly increased compared with the gefitinibtreated group (P < 0.001).
In the middle part of the small intestine, changes were less
pronounced (Fig. 2). The villus height in the gefitinib-treated
animals (0.39 mm) was comparable to controls (0.43 mm).
GLP-2 – treated (0.54 mm) and GLP-2 + gefitinib (0.48 mm) –
treated animals had significantly increased villus height
compared with controls (P < 0.001 and P < 0.05, respectively)
and compared with gefitinib-treated mice (P < 0.001).
In the distal part of the small intestine, treatment with GLP-2
resulted in increased villus height compared with controls
(P < 0.01), gefitinib (P < 0.001), and gefitinib + GLP-2
(P < 0.05; Fig. 2). The villus height in gefitinib- and gefitinib +
GLP-2 – treated animals was comparable to controls.
The depth of the crypts in the small intestine was not
influenced by any of the treatments (Table 2). There was a
tendency toward an increased crypt depth in the proximal and
middle part in the GLP-2 – treated animals; however, this was
not significant (Table 2).
With respect to the large intestine, the four treatment
groups were comparable because no significant differences in
cross-sectional area, crypt depth, and mucin area were seen
(Table 2).
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GLP-2 and Gefinitib-Induced Intestinal Atrophy
Table 2. Morphometric data from mice treated 10 days with PBS, GLP-2, gefitinib, or gefitinib + GLP-2
Gastric mucosa
Mucosal height (mm)
Height of gastric pit (Am)
Mucin area per millimeter mucosa (103 Am2)
Small intestine
Cross-sectional area (mm2)
Proximal
Middle
Distal
Crypt depth (mm)
Proximal
Middle
Distal
Colon
Cross-sectional area (mm2)
Crypt depth (mm)
Mucin area, % of cross-sectional area
PBS
GLP-2
1.32 F 0.09
177 F 17
65.25 F 10.3
1.24 F 0.07
193 F 15
87.65 F 8.5
3.27 F 0.20*
2.50 F 0.22c
1.79 F 0.14
Gefitinib
4.63 F 0.34
3.48 F 0.19
1.95 F 0.09
1.27 F 0.10
162 F 21
65.75 F 8.6
3.40 F 0.15*
2.54 F 0.24c
1.74 F 0.10
Gefitinib + GLP-2
1.16 F 0.05
200 F 9
90.16 F 8.3
4.00 F 0.21
2.83 F 0.22
1.80 F 0.10
0.118 F 0.008
0.113 F 0.005
0.124 F 0.008
0.151 F 0.009
0.123 F 0.006
0.121 F 0.005
0.127 F 0.014
0.110 F 0.004
0.108 F 0.005
0.123 F 0.010
0.115 F 0.007
0.115 F 0.008
2.97 F 0.21
0.033 F 0.002
66 F 7.2
3.66 F 0.22
0.043 F 0.003
58 F 8.2
3.45 F 0.20
0.034 F 0.002
51 F 4.8
2.86 F 0.22c
0.043 F 0.005
62 F 8.6
NOTE: Results are mean F SE.
*P < 0.01 compared with GLP-2 – treated mice.
cP < 0.05.
Discussion
There is increasing evidence that EGFR-TKIs have a place in
the future treatment of different adenocarcinomas. However,
the side effects of blocking the EGF system are undesirable,
especially in cancer patients where the overall health may
already be impaired. The EGFR is present on most epithelial
and stromal cells as well as on some glial and smooth muscle
cells and is essential for normal function and development in
the gut, kidney, urogenital system, and skin (5, 12, 13, 25).
EGFR-inhibiting agents have an advantage over conventional
chemotherapeutic agents in that they selectively block specific
deregulated pathways in tumor cells while having less effects on
normal cell function (5, 26). Thus, the EGFR is an obvious
target in cancer therapy and has already been proven efficient in
clinical studies. However, gastrointestinal side effects need to be
considered. GLP-2 is an intestinal hormone that acts through
not yet settled pathways to induce intestinal growth. Several
studies have shown that the growth factor, EGF and GLP-2,
have similar trophic properties in the gastrointestinal tract
(27 – 36). IGF has recently been suggested to have a crucial role
as mediator of GLP-2 – induced intestinal growth (23), but the
possible role of the EGF system has not been elucidated.
Our hypothesis was that EGFR-TKIs cause atrophy of the
gastrointestinal tract, and unless the EGF system is crucial for
the GLP-2 – mediated stimulation of intestinal growth, treatment with GLP-2 can prevent this atrophy.
Our results show that 10 days of oral treatment with the
EGFR inhibitor gefitinib leads to a decrease in the weight of the
small and large intestines and a decreased absorptive surface
area due to a pronounced atrophy of the villi. EGFR activity
in the small intestinal surface epithelium is almost entirely
restricted to the proliferative crypt region. The receptor is
located on the basolateral surface of the epithelium, and the
role of this growth factor system is probably to stimulate repair
and maintenance of the gut (12, 37 – 40). The reported atrophy
may be due to the absence of these effects on the mucosa
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resulting in reduced mucosal growth. Other consequences from
a lack of this growth factor system have been described by
Troyer et al. in knock-out mice missing some of the EGFR
ligands (amphiregulin, EGF, and transforming growth factor-a).
The duodenum of these animals is prone to spontaneous
ulceration, and the ileal villus height is reduced when compared
with wild-type mice (41). Lesions in the duodenum have also
been reported after treating mice with an EGFR inhibitor (42).
EGFR-/- mice were described by Miettinen et al. (25). These
mice only survive for up to 8 days after birth and suffer from
impaired epithelial development in several organs. The pups
suffer from dehydration and malnutrition and die severely
undernourished. Post mortem findings include hemorrhagic,
distended intestines with a reduced number of shortened villi,
thereby resembling necrotizing enterocolitis (25).
Fig. 2. Villus height in the proximal, middle, and distal part of the small intestine in
PBS, GLP-2, gefitinib, and gefitinib + GLP-2 ^ treated mice. Results are mean + SE.
AA, P < 0.001, compared with gefitinib-treated mice. b, P < 0.05; B, P < 0.01; and
BB, P < 0.001, compared with GLP-2 ^ treated mice. c, P < 0.05, compared with
gefitinib + GLP-2 ^ treated mice.
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Cancer Therapy: Preclinical
Fig. 3. Proximal small intestine stained with PAS-HE showing differences in villus height and crypt depth. A, PBS. B, GLP-2 ^ treated group. C, gefitinib-treated group.
D, GLP-2 + gefitinib ^ treated group. Bar, 100 Am.
These studies underline the importance of the EGF system in
the development, maintenance, and function of the normal gut.
Following treatment with GLP-2, we found increased smalland large-intestinal growth seen as both increased organ weight
and increased villus height. Changes were most pronounced
in the proximal part of the small intestine. When GLP-2 was
given to the gefitinib-treated mice, we found that the gefitinibinduced atrophy could be completely prevented because all
morphometric parameters of the gut were comparable or even
enhanced compared with PBS-treated controls.
The mechanism of GLP-2 – induced growth is still not fully
understood, but the involvement of local growth factors is
probable (24). Recently, the presence of IGF-I was suggested to
be necessary for the GLP-2 intestinal growth response (23).
From the results of the present study, it seems unlikely that
GLP-2 acts via the EGF receptor because we were able to elicit
identical GLP-2 – induced growth responses with or without
concurrent inhibition of the EGFR.
The gastrointestinal side effects observed in patients treated
with gefitinib may be caused by the reduction of the absorptive
surface area resulting from atrophy of the small intestine. In
patients with short-bowel syndrome (where the absorptive
area of the gut is also decreased), GLP-2 injections resulted in
increased intestinal absorptive function, delayed gastric emptying, and a general increase in lean body mass (43). Furthermore, morphometric analysis showed an increase in both
villus height and crypt depth. Compliance in these patients
was excellent, although GLP-2 was given twice daily as s.c.
injections for 35 days (43). A similar improvement of
absorptive function might be observed in patients on gefitinib
treatment also given GLP-2.
The intestinal growth caused by exogenous GLP-2 regresses
to normal after cessation of treatment, indicating that the
epithelial proliferation is dependent on ongoing GLP-2 administration (19). No side effects have been reported after treatment
with GLP-2, which is also in agreement with the assumption
that its only target is the gut (19).
Physiologically, GLP-2 delays gastric emptying (enterogastrone effect; refs. 44, 45), perhaps through inhibiting centrally
induced antral motility (46), thereby acting as one of the
mediators of the so-called ileal brake. The overall combination
of intestinotrophic effects, functional improvement (27 – 30,
33, 34), and an enterogastrone effect makes GLP-2 promising
as an agent for treatment of intestinal insufficiency. These
physiologic properties of GLP-2 would also be of benefit to
cancer patients suffering from diarrhea and weight loss.
Although GLP-2 has proved to be safe in long-term trials
(19, 21), recent studies show that GLP-2 is able to accelerate
growth of chemically induced colonic neoplasms in mice (47).
This effect of GLP-2 is not surprising in view of its trophic
action on the colonic mucosa. On the other hand, GLP-2 alone
has never been found to induce neoplasia. Still, it may be
advisable to perform colonoscopy in patients receiving longterm treatment with GLP-2.
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Clin Cancer Res 2007;13(17) September 1, 2007
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Research.
The Intestinotrophic Peptide, GLP-2, Counteracts Intestinal
Atrophy in Mice Induced by the Epidermal Growth Factor
Receptor Inhibitor, Gefitinib
Kristine Juul Hare, Bolette Hartmann, Hannelouise Kissow, et al.
Clin Cancer Res 2007;13:5170-5175.
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