Hypergastrinemia Promotes Adenoma Progression in the Apcmin؊/؉ Mouse Model of Familial Adenomatous Polyposis1

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[CANCER RESEARCH 61, 625–631, January 15, 2001] Hypergastrinemia Promotes Adenoma Progression in the APCMin؊/؉ Mouse Model of Familial Adenomatous Polyposis1

Sue A. Watson2 and Andrew M. Smith Academic Unit of Cancer Studies, Department of Surgery, University of Nottingham, Nottingham NG7 2UH, United Kingdom

ABSTRACT nary findings of Smith and Watson (18) that human adenomas of all sizes and grades express an isoform of the cholecystokinin B/gastrin Serum hypergastrinemia promotes the growth of colorectal adenocar- receptor. cinoma. Some colorectal adenomas express cholecystokinin B/gastrin re- Thus, the aim of the present study was to evaluate the effect of ceptor mRNA, and thus hypergastrinemia may increase progression through the adenoma-carcinoma sequence. This was investigated in the serum hypergastrinemia induced by omeprazole, a proton pump in- multiple intestinal neoplasia APCMin؊/؉ mouse. Serum gastrin levels in hibitor that increases serum gastrin levels 2–4-fold (11, 12, 19), on APCMin؊/؉ mice were elevated 5–6-fold by oral administration of ome- colonic neoplastic progression in the multiple intestinal neoplasia MinϪ/ϩ prazole (75 mg/kg). Terminal tumor burden was monitored by onset of APC mouse model of APC. anemia. A labeling index was generated by immunohistochemical detection of bromodeoxyuridine incorporation. Serum gastrin was neutralized MATERIALS AND METHODS by antigastrin antibodies raised in situ by use of a gastrin immunogen, Gastrimmune. Hypergastrinemia resulted in reduced survival of the APC- APCMin؊/؉ Mice. APCϪ/ϩ mice were bred within the Cancer Studies Unit ؊ ؉ Min / mice from a median survival of 13 weeks in the controls to 10 weeks at the University of Nottingham under Home Office Project License No. PPL following omeprazole treatment (P < 0.00001, log-rank test). The labeling 4011454. Heterozygous male APCMinϪ/ϩ mice were bred with wild-type indices of adenomas from the small and large intestines of omeprazole- C57/Bl6 female mice. The offspring were characterized for the Min genotype treated mice were increased 35 and 29%, respectively (P < 0.05 and P < by PCR as described previously (20). 0.025, respectively). Gastrimmune immunization reversed both the sur- Entry of Mice into Therapeutic Studies. After results from the PCR vival effect and the increased proliferation resulting from serum hyper- analysis were obtained, APCMinϪ/ϩ-positive mice were randomized into each gastrinemia. Hypergastrinemia may promote the progression of existing experimental group on an ongoing basis because of the limited number of mice premalignant colonic lesions by increasing proliferation. Clinical investi- positive for the Min mutation that are generated from established breeding gations should determine whether this occurs in the human scenario, colonies. Mice were randomized into groups on a litter basis with unequal considering the widespread use of proton pump inhibitors. numbers being a result of the distribution of Min positive and negative mice. Experiment 1: Effect of Omeprazole-induced Hypergastrinemia on Adenoma Number and Size and Animal Survival. An initial study was INTRODUCTION performed to determine the effect of hypergastrinemia on tumor burden and survival of APCMinϪ/ϩ mice. APCMinϪ/ϩ mice 4 weeks of age were recruited Gastrin has been described as a growth factor for colorectal malig- to the following groups: group 1, oral vehicle for omeprazole [PBS (pH 7.2)]; nancy for well over a decade, and it is known to mediate proliferation group 2, omeprazole (75 mg/kg in a single oral dose; n ϭ 10). via both endocrine and autocrine/paracrine mechanisms (1–3). Tumor size was measured by image analysis by use of Leica Qwinn image Elevated serum gastrin levels can be a feature of a number of processing and analysis system run on a Leica Q5001W PC. clinical conditions, including ZES3 (4–7) and PA (8), but possibly Experiment 2: Effect of Omeprazole-induced Hypergastrinemia on more importantly as a result of infection with Helicobacter pylori (9, Survival with or without Immunization with the Gastrin Immunogen, Gastrimmune. APCMinϪ/ϩ mice 4 weeks of age were recruited to the fol- 10) and after the administration of proton pump inhibitors (11, 12). lowing groups: group 1, oral vehicle for omeprazole ϩ control immunogen In humans, increased proliferation of colonic crypts has been con- (n ϭ 20); group 2, omeprazole (75 mg/kg in a single oral dose) ϩ control firmed in patients with PA and ZES (13). However, the majority of immunogen (n ϭ 20); group 3, omeprazole ϩ Gastrimmune (n ϭ 16). epidemiological studies performed to date in patients with sustained Gastrimmune is a gastrin immunogen composed of the NH2-terminal nine hypergastrinemia have failed to show an enhanced risk of colorectal amino acids of gastrin-17 linked via a peptide spacer to diphtheria toxoid. cancer. In patients with PA, although conflicting studies have been Treatment with Gastrimmune generates antibodies that neutralize the amidated reported, the majority show that colorectal cancer incidence is not and glycine-extended forms of gastrin-17 (Aphton Corporation, CA; Ref. 21). increased (14, 15). This has also been shown in patients with ZES A rat-specific form of the immunogen was formulated at 2 mg/ml and injected (16). This failure may be attributed to lack of control of confounding s.c. into mice in 100-␮l volumes at week 4, and at three-week intervals factors, such as H. pylori in the control groups. A recent study, in thereafter. The rat sequence used within the immunogen differs only by one amino acid from the mouse sequence, and antibodies raised by rat Gastrim- which H. pylori infection was controlled for, showed that hypergas- mune previously have been shown to neutralize mouse gastrin (data not trinemia is associated with a 3.3-fold increase in the relative risk of shown). Control mice receiving immunogen constituents only were given the developing colorectal cancer (17). It is conceivable, therefore, that formulation without the active peptide. hypergastrinemia may promote progression through the adenoma- Experiment 3: Effect of Preimmunization with Gastrimmune Immuno- carcinoma sequence. This hypothesis is strengthened by the prelimi- gen on the Survival Effect Observed with Omeprazole Treatment. APC- MinϪ/ϩ mice 4 weeks of age were recruited to the following groups: group 1, ϩ ϭ Received 3/27/00; accepted 11/6/00. vehicle control immunogen (n 22 mice); group 2, omeprazole (75 The costs of publication of this article were defrayed in part by the payment of page mg/kg) ϩ control immunogen (n ϭ 30 mice); group 3, omeprazole ϩ Gastrim- charges. This article must therefore be hereby marked advertisement in accordance with mune (n ϭ 30 mice); group 4, vehicle ϩ Gastrimmune (n ϭ 18 mice). 18 U.S.C. Section 1734 solely to indicate this fact. Gastrimmune was injected 2 weeks prior to omeprazole. (The latter was 1 This work was supported by the Aphton Corporation, Woodland, CA. 2 To whom requests for reprints should be addressed, at Cancer Studies Unit, D Floor, administered from week 6 when antibodies against Gastrimmune were known West Block, QMC University Hospital, Nottingham NG7 2UH, United Kingdom. Phone: to be detectable.) Fax: 44-115-9709902; E-mail: [email protected]. Termination of APCMin؊/؉ Mouse Experiments. At the end-stage of ;44-115-9709248 3 The abbreviations used are: ZES, Zollinger Ellison syndrome; PA, pernicious ane- their disease, APCMinϪ/ϩ mice bleed from their multiple polyps, giving rise to mia; APC, adenomatous polyposis coli; Min, multiple intestinal neoplasia; GI, gastroin- testinal; BrdUrd, bromodeoxyuridine; CCKBR, cholecystokinin B receptor; GAPDH, anemia, which was initially confirmed by hematocrit measurements [median glyceraldehyde-3-phosphate dehydrogenase; LI, labeling index. hematocrit, 0.136 (range, 0.058–0.153) compared with 0.385 (range, 0.381– 625

0.391] in wild-type C57/Bl6 mice. Twelve days after the onset of anemia, mice triphosphates, 20 ␮M each primer, and 1 unit of AmpliTaq Gold (PE Applied were terminally anesthetized by a 300-␮l injection containing 0.315 ng/ml Biosystems). The PCR cycle consisted of a 95°C activation stage for 10 min Hypnorm and 5 ng/ml Hypnovel (Jannsen-Wickham, Bucks, United Kingdom, followed by 40 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 30 s. After and Roche-Lewis, East Sussex, United Kingdom) by an experienced, inde- a final extension stage of 72°C for 10 min, the PCR products were separated pendent observer who was blinded to the treatment groups. Anemia previously on a 2% (w/v) agarose gel and stained with ethidium bromide. The mouse has been validated as a consistent end point for APCMinϪ/ϩ mouse termination colon cancer cell line MC26 was used as a positive control. A negative control (20). After termination, the GI tracts of the APCMinϪ/ϩ mice were removed, was also included that was not reverse transcribed and confirmed the absence and the numbers of visible tumor nodules in both the small and large bowel of genomic DNA. were counted with the aid of a dissecting microscope. The mucosa was fixed Real-Time PCR to Quantify CCKBR Expression. RNA was extracted in formal calcium (40% formaldehyde, 10% CaCl2 in distilled water) prior to from tissue and reverse transcribed from random hexamer primers (Pharmacia) embedding in paraffin wax for histological analysis. using Supercript reverse transcriptase (Life Technologies, United Kingdom). The United Kingdom Coordinating Committee for Cancer Research Guide- Real-time PCR was performed using the 5700 Sequence Detection System (PE lines were adhered to throughout all animal experimentation. Applied Biosystems, Warrington, United Kingdom). Each PCR was performed Measurement of the Proliferation Index of the GI Mucosa. One h before according to manufacturer’s instructions using 1 ␮l of sample cDNA in a 25-␮l termination, mice received i.p. injections of 10 mg/kg BrdUrd (Sigma). After reaction volume. The reaction buffer was prepared from the SYBR Green PCR paraffin-embedding, 4-␮m sections were stained with H&E to confirm correct Core Kit (PE Applied Biosystems) and consisted of 1ϫ SYBR Green PCR orientation of the crypts and with an antibody directed against BrdUrd (mouse Buffer (PE Applied Biosystems), 3 mM MgCl2, 0.2 mM dATP, 0.2 mM dCTP, monoclonal, used at 1:500 dilution; Dakopatts). 0.2 mM dGTP, 0.4 mM dUTP, 0.25 units of AmpErase UNG (PE Applied Two experienced observers, blinded to the treatment groups, counted the Biosystems), and 0.625 units of AmpliTaq Gold (PE Applied Biosystems). The number of positive nuclei in the crypts from both the small and large intestine. murine CCKBR primers MCK1 and MCK2 and the GAPDH primers (GAPU, Ten whole crypts from different areas of the colonic mucosa and the small 5Ј-GGTGAAGGTCGGAGTCAACGGA-3Ј; GAPL, 5Ј-GAGGGATCTCGC- intestinal mucosa of each mouse were identified where possible. The number TCCTGGAAGA-3Ј) were included in parallel reactions at a final concentra- of total positive nuclei within the epithelial cells was counted, together with the tion of 100 nM each. The fluorescence of the SYBR Green dye bound to the total number of cells per crypt. This was repeated with sections from 4–11 CCKBR and GAPDH PCR products was measured after each cycle by the 5700 animals. The total number of cells per crypt in the large or small intestine were System, and the cycle number was recorded when the accumulated signal consistent both within and between experimental animals. crossed an arbitrary threshold (Ct value). The relative gene expression for each Ϫ For scoring of the polyps, each section from each mouse was cut at the same sample was determined using the formula 2Ct(GAPDH) Ct(CCKBR), and reflected depth (2 mm), and each polyp was analyzed by choosing a representative CCKBR gene expression normalized to GAPDH levels. low-power field and by using image analysis to assess the area of BrdUrd Measurement of Serum Omeprazole Levels. Chilled methanol (0.4 ml) staining as a percentage of the total tumor tissue. For assessment of the was added to 0.1 ml of serum. After mixing, samples were centrifuged at proliferation of adenoma and carcinoma samples that were taken from the large 2000 ϫ g for 10 min at 4°C. The supernatant was then analyzed by high- and small intestines, image analysis was performed by the use of the Leica performance liquid chromatography together with standard samples prepared Qwin image processing and analysis system. BrdUrd staining was specifically by adding known amounts of omeprazole to control serum. The high-perfor- measured and expressed as a function of total tumor tissue area. Intraobserver mance liquid chromatography chromatographic conditions were as follows: variation, in which different fields of the same section were assessed, was Prodigy OGS (250 ϫ 4.5 mm) column; mobile phase, 42% acetonitrile in 7.5 5.6%, and interobserver variation was 7.5%. mM diammonium hydrogen phosphate (Sigma); flow rate, 1.0 ml/min; UV Histological Assessment. The GI tracts from end-stage APCMinϪ/ϩ mice detection at 304 nm; injection volume, 0.05 ml; analysis time, 30 min; treated with either vehicle or omeprazole, as detailed in “Experiment 2,” were detection time, 6.3 min. The linearity of the standard curve was confirmed over fixed in formal calcium, processed into paraffin, and stained with H&E. A a sample concentration of 1–20 ␮g/ml (r ϭ 0.99). consultant pathologist assessed the sections in a blinded manner detailing Measurement of Antibodies Raised by Gastrimmune. The measurement architectural detail and degree of dysplasia as well as necrotic and vascular of antibodies raised against Gastrimmune was performed according to a appearance. method described previously (21). RIA to Determine Serum Gastrin Levels. Prior to termination of the Statistical Assessment of Results. Survival analysis was performed by a APCMinϪ/ϩ mice, the animals were fasted overnight. Blood was collected by log-rank test using a multiple group comparison, and the in vitro results were cardiac puncture, and serum was prepared by centrifugation after clotting at compared using a one-way ANOVA, a Student’s t test, or the Mann-Whitney 4°C. The serum concentrations of total amidated gastrin were measured using test with the Minitab statistical package. the L2 antiserum and also antiserum directed against progastrin and glycine- extended gastrin, as described previously (22), or levels were measured by the RESULTS use of an ELISA-based kit (Invitrogen, Newcastle, United Kingdom). To measure “antibody-bound” serum gastrin concentrations in mice immu- Omeprazole Induction of Serum Hypergastrinemia. Elevation nized with Gastrimmune, serum was collected as described above. The anti- in serum gastrin concentrations was achieved by administration of the body:gastrin complexes were immunoprecipitated using newborn calf serum proton pump inhibitor omeprazole with the dose chosen so that it (Sigma) and 25% polyethylene glycol (8000; Sigma), with a final newborn calf serum-polyethylene glycol-test serum ratio of 1:4:5. The mixture was vor- reflected the elevation of serum gastrin seen in patients on a mainte- texed, and precipitates were pelleted by centrifugation at 460 ϫ g for 30 min nance dose of omeprazole (80 pM; Refs. 15, 16). In an initial pilot at 4°C. The precipitate was then resuspended in 200 ␮l of 0.02 M veronal study, mice were dosed with 12.5, 37.5, and 75.0 mg/kg of omepra- buffer (pH 8.4; 4.12 g/liter sodium barbitone, 0.5 g/liter sodium azide, 1.65 zole daily by oral gavage for a period of 7 days and were fasted prior g/liter serum BSA; all reagents from Sigma). The tubes containing the recov- to cardiac puncture. Mice treated with vehicle had a median serum ered antibody:antigen complexes were then tightly capped and placed in a total amidated gastrin concentration of 15.5 pM. When omeprazole boiling water bath for 5 min. Recovered gastrin was then measured from a was dosed at 12.5 mg/kg, the median serum amidated gastrin concen- standard gastrin-17 curve prepared with charcoal-stripped pooled mouse trations increased to 36.5 pM compared with medians of 52.5 and serum. 102.0 pM in mice treated with omeprazole doses of 37.5 and 75 mg/kg, Reverse Transcription-PCR to Confirm CCKBR Expression. Mouse- respectively. In the group treated with 37.5 mg/kg, 4 of 16 mice specific primers were designed to assess classical CCKBR. The primers MCK1 (5Ј-CCCTCCTCAACAGCAGTAGC-3Ј) and MCK2 (5Ј-GGGTGATTC- achieved a serum amidated gastrin concentration of 80.0 pM or greater GAATGGTCAAC-3Ј), specific for exons 1 and 2, respectively, were used to compared with 12 of 16 in the 75 mg/kg omeprazole-treated group. amplify cDNA prepared from tumor and nonmalignant mucosa from both large The highest dose of omeprazole was therefore chosen for the in vivo and small intestine. The 50-␮l PCR reactions were set up using 3 ␮l of sample studies, which generated median serum omeprazole levels of 5.82 cDNA in 1ϫ PCR Buffer (PE Applied Biosystems), 40 ␮M deoxynucleotide ␮g/ml (interquartile range, 1.77–17.3; n ϭ 10 mice). 626

No glycine-extended gastrin or progastrin was detected by RIA in either C57/Bl6 or Balb/C mouse strains after omeprazole treatment. Effect of Omeprazole-induced Hypergastrinemia on Tumor Burden and Survival of APCMin؊/؉ Mice. The results are shown in Table 1. There was no increase in either small or large intestinal tumor number at termination (Table 1). However, when mean total tumor size (cumulative size of large and small tumors within either the small or large intestine) was assessed, there was a significant increase in large (2.3-fold; P Ͻ 0.05) but not small intestinal tumor load. Survival was significantly reduced in the omeprazole group (mean survival of 9.6 weeks compared with 13.9 weeks in the vehicle control-treated group; P Ͻ 0.0001). Tumor burden/time also revealed significant differences between both the small and large intestine (1.46- and 2.6-fold increases, respectively, in the omeprazole-treated groups; P Ͻ 0.025 for both). -Gastrimmune Immunization of APCMin؊/؉ Mice. The first ther apy study involved immunization with Gastrimmune at the same time as treatment with omeprazole (both at week 4). An initial study was Fig. 1. Effect of Gastrimmune-omeprazole combinations on the survival of APCMinϪ/ϩ F ϭ performed to determine the time of onset of antigastrin antibody titers mice. Group 1 ( ), NaHCO2 together with immunogen constituents (n 20); group 2 (E), omeprazole ϩ immunogen constituents (n ϭ 16); group 3 (), omepra- induced by Gastrimmune immunization, and by day 14, antibodies zole ϩ Gastrimmune (n ϭ 20). Log-rank analysis showed that the survival of omeprazole- were measurable in the serum (data not shown). treated mice was significantly reduced compared with the vehicle control (P ϭ 0.00001) ϩ ϭ Omeprazole, together with administration of the control immuno- and the omeprazole Gastrimmune-treated group (P 0.0002). gen, was shown to significantly reduce the survival of APCMinϪ/ϩ mice compared with the vehicle control (P ϭ 0.00001, log-rank test) with a median 50% survival of 10 weeks compared with 13 weeks sera as detailed in “Materials and Methods.” Animals receiving (Fig. 1). When Gastrimmune immunization was combined with ome- Gastrimmune ϩ vehicle had a mean bound gastrin level of 18.35 prazole treatment, there was a increase in the median 50% survival to pmol/ml (SD, 2.32 pmol/ml), which approximates the fasting serum 14 weeks, which was significantly different from omeprazole alone gastrin levels measured previously. In the Gastrimmune ϩ omepra- (P ϭ 0.0002, log-rank test) but not significantly different from the zole-treated mice, the bound serum gastrin levels had increased to vehicle control (P ϭ 0.2602, log-rank test). 55.6 pmol/ml (SD, 9.65 pmol/ml), a 3-fold significant increase over In the second study, mice were preimmunized with Gastrimmune at the levels shown in the Gastrimmune ϩ vehicle-treated mice week 4, and omeprazole treatment was delayed until week 6. This (P Ͻ 0.0001). allowed Gastrimmune-induced antibodies to be generated before se- To determine adenoma proliferation at earlier stages of disease, a rum hypergastrinemia was initiated. Fig. 2 shows the results of this third study was initiated in which APCMinϪ/ϩ mice were treated with study. Omeprazole significantly reduced survival (P ϭ 0.0038, log- the same treatment regimes as study 2 but sacrificed at week 12. The rank test), with a median 50% survival of 11 weeks, despite a number of tumors macroscopically visible in the small and large treatment delay. The vehicle group had a median 50% survival of 13 bowel was counted and the LI measured. weeks, as did the group receiving the Gastrimmune-omeprazole com- Measurement of small intestinal tumors numbers revealed no bination, and there was no significant difference between these two significant difference between any of the groups. With large in- groups (P ϭ 0.1103, log-rank test). However, the survival of the testinal tumor number, there was a 2.5-fold increase, from 0.88 in group treated with the Gastrimmune-omeprazole combination was the vehicle control-treated group to 2.22 in the omeprazole-treated significantly increased from the group treated with omeprazole alone group (P ϭ 0.030). This appeared to be partially reduced when (P ϭ 0.0002, log-rank test). Gastrimmune alone was also shown to omeprazole was combined with Gastrimmune to give a mean significantly increase survival when compared with the vehicle con- number of 1.65, which was not significantly different from the trol (P ϭ 0.0017) with a 50% median survival of 19 weeks. vehicle control (P ϭ 0.075). The group treated with Gastrimmune In the Gastrimmune ϩ vehicle-treated group, terminal antibody alone had a mean large intestinal tumor number of 0.76, a 14% levels had a mean absorbance of 0.447 (SD, 0.43) compared with a reduction, which again was not significantly different from the mean of 0.330 (SD, 0.016) for the mice treated with Gastrim- vehicle control. mune ϩ omeprazole, a 26% reduction, which was significant The LI as measured by BrdUrd uptake from the same study is (P Ͻ 0.02, Student’s t test). Animals treated with either vehicle or shown in Table 1. Omeprazole treatment significantly increased the LI omeprazole only had no detectable antibody-specific absorbance. of normal large intestine (53%; P Ͻ 0.020) and large intestinal tumors Antibody-bound gastrin levels were measured after processing of (29%; P Ͻ 0.025) compared with the vehicle control. Histological

Ϫ ϩ Table 1 Effect of omeprazole-induced hypergastrinemia on tumor burden and survival of APCMin / mice Results are shown as the mean (SD); n ϭ 10 mice per group. Mean tumor no. Mean total tumor size (mm2) Tumor burden/time (mm2/week) Mean Group Largeb Small Large Small survival (weeks) Large Small Vehicle control 0.90 (0.87) 58.3 (17.3) 0.87 (0.82) 29.3 (9.4) 13.9 (1.72) 0.08 (0.055) 2.03 (0.60) Omeprazole 1.90c (1.96) 55.8c (14.1) 2.0d (1.76) 27.4c (8.2) 9.6f (1.9) 0.21e (0.17) 2.97e (1.12) b Large, large intestine; small, small intestine. c–e Student’s t test: c nonsignificant; d P Ͻ 0.05; e P Ͻ 0.025. f Log-rank test: P Ͻ 0.0001. 627

intestinal crypts from vehicle-treated APCMinϪ/ϩ mice (Fig. 3A). There was also an obvious increase in BrdUrd incorporation in large intestinal adenomas/carcinomas. Fig. 3B shows a polypoid carcinoma from a control mouse and an omeprazole-treated mouse at the end- stage of disease progression. The LI of the small intestinal normal mucosa (from lower and upper regions) was not significantly affected by omeprazole treatment, whereas that of small intestinal tumors was increased (35% increase; P Ͻ 0.05). Gastrimmune treatment significantly reduced the LI of the normal large intestinal mucosa (20% reduction; P Ͻ 0.050) and large intestinal tumors (42% reduction; P Ͻ 0.002). Gastrimmune had no effect on the growth of normal small intestinal mucosa, but significantly reduced the growth of tumors from the small intestine (22% reduction; P Ͻ 0.025). Gastrimmune and omeprazole in combination significantly inhibited the LI increase induced by omeprazole treatment in all tissues responding to omeprazole (significance levels for normal and malig- Fig. 2. Effect of Gastrimmune preimmunization on survival of APCMinϪ/ϩ mice treated with omeprazole. Group 1 (F), vehicle ϩ control immunogen (n ϭ 22); group 2 nant large intestinal mucosa and small intestinal tumors are shown in (E), omeprazole ϩ control immunogen (n ϭ 30); group 3 (), omeprazole ϩ Gastrim- Table 2). ϭ ƒ ϩ ϭ mune (n 30); group 4 ( ), vehicle Gastrimmune (n 18). Omeprazole significantly When Gastrimmune and omeprazole were coadministered, the LI reduced survival (P ϭ 0.0038, log-rank test). Survival of the group treated with Gastrim- mune ϩ omeprazole was significantly increased from the group treated with Omeprazole was significantly reduced compared with the vehicle control in large alone (P ϭ 0.0002), and treatment with Gastrimmune alone also had significantly intestinal normal and malignant mucosa and in small intestinal normal increased survival compared with the control (P ϭ 0.0017). but not malignant mucosa (significance levels shown in Table 2). In most groups in which Gastrimmune significantly inhibited the assessment of the large intestinal crypts after omeprazole treatment mucosal LI, the difference was nonsignificant when compared with revealed a distortion in their shape attributable to the increased pro- the respective Gastrimmune-omeprazole combination. However, in liferation together with an increase of the proliferative zone toward large normal intestinal mucosa, there was a significant 30% reduction the top of the crypt (Fig. 3A). This was not observed in the large in the LI after Gastrimmune-omeprazole treatment compared with the

Fig. 3. Histological effect of omeprazole on BrdUrd incorporation of intestinal normal mucosa and adenomas from the APCMinϪ/ϩ mice. A, normal large intestinal mucosa of a control mouse and a omeprazole-treated mouse (ϫ1000 magnification). B, polypoid carcinomas from the large intestine of a control and a omeprazole- treated mouse (ϫ250 magnification). H&E-stained sections from paraffin-embedded formalin-fixed samples.

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Table 2 Effect of Gastrimmune with or without omeprazole on the LIsa of mucosal Ϫ ϩ DISCUSSION samples in APCMin / mice (mean LI Ϯ SD)b Large intestine Small intestine The role of serum hypergastrinemia in colorectal carcinogenesis remains controversial; most epidemiological studies show that hyper- Group Normal Tumor Normal Tumor gastrinemia does not increase colonic tumor incidence (14–16), but Ϯ Ϯ Ϯ Ϯ Vehicle control 4.9 2.1 13.2 7.4 6.5 2.8 12.1 7.9 does increase colonic mucosal proliferation (13). Past studies evalu- Gastrimmune 3.9 Ϯ 2.8 7.7 Ϯ 5.4 4.8 Ϯ 1.4 9.4 Ϯ 8.0 Omeprazole 7.5 Ϯ 3.8 17.0 Ϯ 8.3 6.2 Ϯ 1.8 16.3 Ϯ 11.8 ating the effect of hypergastrinemia on tumor incidence in humans did Gastrimmune ϩ omeprazole 2.7 Ϯ 2.5 8.1 Ϯ 4.9 5.1 Ϯ 1.1 9.9 Ϯ 6.2 not control for confounding factors such as H. pylori in the control Statistics (one-way ANOVA) population. In a recent study in which H. pylori infection was taken Normal large intestinal mucosa into account, a temporal relationship between elevated gastrin levels Vehicle control vs. Gastrimmune 20% reduction P Ͻ 0.050 and development of colorectal malignancy was shown (17). Vehicle control vs. omeprazole 53% increase P Ͻ 0.020 Gast/Omepc vs. vehicle control 45% reduction P Ͻ 0.0001 What is not known is how hypergastrinemia affects premalignant Gast/Omep vs. omeprazole 64% reduction P Ͻ 0.0001 change in the colon. This question was addressed in the present study Gast/Omep vs. Gastrimmune 30% reduction P Ͻ 0.010 by use of the APCMinϪ/ϩ mouse model of familial adenomatous Large intestine tumors Vehicle control vs. Gastrimmune 42% reduction P Ͻ 0.002 polyposis. A state of hypergastrinemia was induced by the proton Vehicle control vs. omeprazole 29% increase P Ͻ 0.025 pump inhibitor, omeprazole, such that serum gastrin concentrations in Ͻ Gast/Omep vs. vehicle control 38% reduction P 0.0001 the mice were elevated to the same level as patients on a maintenance Gast/Omep vs. omeprazole 52% reduction P Ͻ 0.0001 Normal small intestinal mucosa dose of omeprazole (23, 24). The variations in serum gastrin concen- Gast/Omep vs. vehicle control 23% reduction P Ͻ 0.01 trations were large and may have included a postprandial element, Small intestine tumors Vehicle control vs. Gastrimmune 22.3% reduction P Ͻ 0.025 despite fasting, because mice are known to be coprophagic. The high Vehicle control vs. omeprazole 35% increase P Ͻ 0.050 omeprazole dose may have been necessary because, unlike rats, which Gast/Omep vs. omeprazole 39% reduction P Ͻ 0.005 are acutely sensitive to omeprazole in terms of serum gastrin eleva- a The LIs of normal colonic and small intestinal crypts were assessed by BrdUrd tion, mice are more refractory, which may relate to enterochromaffin- incorporation and immunohistological assessment (positive nucleii were counted by two independent observers). The LIs of the tumors were measured by image analysis. like cell density and differences in omeprazole metabolism (25). b Each mean was derived from 20–60 observations from 4–11 mice. c Gast/Omep, Gastrimmune/omeprazole.

LI in the mucosa of the group treated with Gastrimmune alone (P Ͻ 0.010). Histological Evaluation of Intestinal Mucosa. Polyps in both vehicle control- and omeprazole-treated groups showed moderate dysplasia without significant pseudostratification of epithelial cells. Small polyps involved few glands and appeared as sessile tubular adenomas, with very little villous formation. As adenomas enlarged, they became polypoid with surface ulceration and vascular stroma. Subtle changes in dysplasia between adenomas was difficult to quantify in an objective manner. However, no gross changes in dysplasia were evident between the two groups. CCKBR mRNA Expression in the APCMin؊/؉ Mouse Intestinal Mucosa. CCKBR was expressed at the gene level on polyps from both the small and large intestine. Fig. 4 shows a gel in which a specific 99-bp band is shown for the positive control cell line, MC26, together with polyps and normal mucosa from the large and small intestines. CCKBR mRNA levels were measured in both large and Fig. 5. CCKBR gene expression of small/large intestinal polyps after treatment with small intestinal polyps with and without treatment with omeprazole. omeprazole (75 mg/kg). Gene expression of mouse CCKBR was assessed by real-time PCR after preparation of cDNA from total RNA that had been extracted from snap-frozen There was a significant 6-fold increase in CCKBR mRNA levels in tissue. CCKBR mRNA levels were expressed as a function of the housekeeping gene, .P ϭ 0.0094, Mann-Whitney test ,ء .hypergastrinemic mice (P Ͻ 0.001; Fig. 5). GAPDH. n ϭ 12 measurements/group

Fig. 4. Southern blot showing CCKBR expression by APCMinϪ/ϩ mouse normal mucosa and adenomas from both the small and large intestine. Lanes 1 and 2, reverse transcriptase-negative controls; Lane 3, MC26; Lanes 4 and 9, molecular weight markers (␾X174 HaeIII digest); Lanes 5–8, large intestine polyps; Lanes 10 and 11, small intestine polyps; Lane 12, normal large intestine mucosa; Lane 13, normal small intestine mucosa. Re- verse transcription-PCR was performed on cDNA prepared from extracted RNA using primers specific for mouse CCKBR. The product generated was 99 bp.

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Omeprazole-induced hypergastrinemia significantly decreased the sponded in an endocrine manner to basal gastrin levels. Antibody- survival of APCMinϪ/ϩ mice. Examination of the normal and malig- bound gastrin was indeed confirmed to be present in normogastrine- nant mucosa from both the small and large bowels of these mice mic APCMinϪ/ϩ mice. It previously has been shown that the gastrin revealed an enhanced proliferation in the tumors from both areas. gene is activated in normal mucosa and tumors in the APCMinϪ/ϩ Thus, omeprazole appeared to increase proliferation of the tumors mouse model and that gastrin immunoreactivity can be detected (31). arising within the GI mucosa, which caused the terminal stage of the If the gastrin is increasing proliferation in an autocrine manner, this disease to be reached at an earlier time point. This was confirmed in may partially explain the increased LI in the normal GI mucosa of the first experiment when tumor burden/time was significantly en- APCMinϪ/ϩ mice compared with Min-negative mice (31). Gastrin hanced in both small and large intestinal adenomas from omeprazole- gene expression and progastrin and glycine-extended gastrin previ- treated mice. In mice terminated at 12 weeks of age, omeprazole had ously have been shown to be expressed in polyps arising within both increased the macroscopic tumor number 2.5-fold. This is possibly a small and large intestinal mucosa of mice with a mutated APC gene result of enhanced proliferation, resulting in earlier visualization of (29, 32). Because antibodies raised by Gastrimmune neutralize gly- the tumors rather than an effect on tumor incidence. Histological cine-extended G17, the antibodies may therefore partially inhibit evaluation of the adenomas revealed that smaller lesions were sessile autocrine pathways established by the tumors. This is relevant because tubular adenomas, becoming polypoid with increasing size. All a number of recent publications have shown that in mice in which showed moderate dysplasia, and after treatment with omeprazole, there is overexpression of precursor gastrin molecules, there is a there was no gross change in the dysplasia exhibited by the lesions. predisposition to aberrant colonic crypt foci and adenomas and car- Thus, in the time frame of these experiments, an increase in prolifer- cinomas in response to a chemical carcinogen (31, 33). ation rather than malignant potential was observed. In conclusion, gastrin may enhance progression to malignancy In the present study, large but not small bowel mucosal crypts were when premalignant changes have occurred in the large and small shown to respond proliferatively in omeprazole-treated mice, which intestines. The findings of these studies highlight an urgent need to may reflect the differential sensitivity that the GI tract has to gastrin investigate the possibility that gastrin may increase progression of peptides (26). In the study by Thorburn et al. (17), the association existing premalignant colorectal lesions in a clinical setting. between gastrin and malignancy was more closely associated in rectal cancer, which lends support to the finding that gastrin has a greater ACKNOWLEDGMENTS proliferative role in the distal GI tract. In the present study, the large intestinal crypts from omeprazole-treated APCMinϪ/ϩ mice had a We would like to acknowledge T. Morris, Unit Manager, Academic Unit of Cancer Studies, for performing the in vivo studies, Dr. P. D. James, Patholo- distorted structure and an increase in the proliferative zone from the MinϪ/ϩ lower third to the whole length of the crypt. This has been shown in gist, for histological evaluations of the APC intestinal sections, Dr. D. McWilliams for performing the molecular biology on the APCMinϪ/ϩ mouse patients with an increased risk of colon cancer (27, 28). samples, and P. Clarke for performing the image analysis and the BrdUrd The effect on survival was confirmed as gastrin-mediated by use of the staining. gastrin immunogen, Gastrimmune, which produces antibodies that neutralize both amidated and glycine-extended gastrin-17 with high affinity REFERENCES (21). Antibodies raised by Gastrimmune do not bind cholecystokinin or other gastrin species such as G34 and pentagastrin. Antigastrin anti- 1. Watson, S. A., Durrant, L. G., Crosbie, J. D., and Morris, D. L. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. Hypergastrinemia Promotes Adenoma Progression in the APC Min−/+ Mouse Model of Familial Adenomatous Polyposis

Sue A. Watson and Andrew M. Smith

Cancer Res 2001;61:625-631.

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