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Carcinogenesis |
University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin 53792 [R. F. J., C. E. C., K. T., M. A. N.], and Chemoprevention Branch, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 20892 [G. K., E. T. H., R. A. L.]
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ABSTRACT |
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Piroxicam at 12, 25, and 50 ppm in the diet caused dose-dependent decreases in the number of tumors in the middle and distal portions of the small intestine. This decrease in tumor multiplicity was associated with a striking decrease in the size of those tumors that did grow out. In contrast, none of the doses of piroxicam alone decreased tumor multiplicity in the proximal portion of the intestine (duodenum). Exposure to DFMO (0.5 or 1.0% in water) caused a dose-dependent decrease in tumor multiplicity in the middle and distal portions of the small intestine. However, this decreased multiplicity was not associated with a striking decrease in the size of the tumors. Combined treatment of mice with piroxicam plus DFMO was much more effective than either agent alone and resulted in a significant number of mice totally free of any intestinal adenomas (P < 0.001), in contrast to the 100% incidence and high multiplicity in control Min mice. In addition to this profound effectiveness in reducing tumor number, the few residual tumors in mice treated with the combined drugs were markedly smaller in size than tumors that arose from control Min mice. These experiments suggest that selective COX-2 inhibition combined with ODC inhibition is a very promising approach for colon cancer prevention.
These COX-2 and ODC inhibitor drugs were not overtly toxic at the doses
used when administered to mice after weaning. However, when treatment
was begun in utero, the Mendelian expected progeny ratio
of 1:1 that we routinely obtained in untreated control litters
was no longer observed. ApcMin/+ progeny
of pregnant dams treated with piroxicam and/or DFMO were reduced in
number and their ratio to Apc+/+ progeny was
decreased to 
0.28:1. Thus, these agents are effective against
adenomas that have homozygous mutation of the APC gene and
also select against fetuses bearing a heterozygous mutation in the
APC gene.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another specific chemical that demonstrates significant promise in preventing colon cancer is DFMO. DFMO is a highly specific suicide inhibitor and substrate analogue for the enzyme ODC (17 , 18) , which catalyzes the conversion of ornithine to putrescine. ODC is overexpressed in a wide variety of invasive and preinvasive neoplastic lesions (19) . In fact, this enzyme may act as an oncogene inasmuch as transgenic mice overexpressing ODC have been shown to develop skin cancer (14) . DFMO has previously been shown to be highly effective as a preventive agent against azoxymethane induced colon cancer in rats (1 , 20) , and is presently undergoing Phase II clinical trials in humans (21 , 22) .
Although highly effective, certain of the NSAIDS cause untoward effects, particularly GI problems. Hundreds of thousands of emergency hospital visits and thousands of deaths per year are associated with this class of agents (23) . Regarding colon cancer prevention a number of potential solutions to GI side-effects arise: (a) the use of highly specific COX-2 inhibitors that do not exhibit the GI problems associated with COX-1 inhibitors (24, 25, 26) ; or (b) the potential use at lower doses of two different classes of agents—doses at which these agents do not exhibit significant adverse side effects. This latter approach of combining agents with nonoverlapping mechanisms and nonoverlapping toxicities has been routinely used in chemotherapy. However, it has also been applied in preclinical chemoprevention studies on both mammary and colon tumors (3 , 25 , 27 , 28) .
In the present study, we have examined the chemopreventive efficacy of DFMO, or piroxicam alone, or the combination of the two agents to prevent the development of adenomatous lesions in heterozygous Min mice that bear a mutation in codon 850 of the Apc gene (29) . As a portion of this experiment, we administered piroxicam or DFMO in utero. Interestingly—although the drugs had embryotoxic properties against all of the genotypes—at these doses we observed preferential toxicity against those with Apc mutation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genotyping.
The presence of the mutant allele was detected in DNA extracted from
blood or skin using an allele-specific PCR assay as described
previously (6)
. Briefly, an oligonucleotide primer
(Apc-mutant) was designed so that the Min
mutation [which converts codon 850 from a leucine (TTG) to amber
(TAG)] is complementary to the 3' end of the primer and is, therefore,
amplifiable, but the noncomplementary wild-type sequence does not
amplify (6)
. An internal control was provided by a second
primer at a location at which wild type and mutant do not differ. This
optimized PCR technique correctly identified the genotype in 99.8% of
assays as verified by repeat analysis performed whenever the phenotype
seemed discordant with the presumed genotype (the presence of adenomas
in a presumed +/+ mouse or their complete lack in a presumed
Min/+ mouse).
Drug Treatment.
Piroxicam (CAS 36322-90-4) was purchased from Sigma Chemical
Company (St. Louis, MO) and DFMO (CAS 70052-12-9) was a gift from Dr.
Ajit Verma at the University of Wisconsin (Madison, WI). For the
standard treatment groups (n = 144 total, 16
per group), piroxicam at the intended concentration was mixed in the
diet beginning at approximately age 30 days; mice were killed at age 90
days after 2 months of treatment. Mice treated with DFMO were given
water mixed with the intended concentration of that drug from age 30 to
90 days. For the in utero treatment groups
(n = 120 total, 30 per group), exposure to
DFMO and/or piroxicam began at gestational age zero, indirectly via
treatment of their mothers throughout pregnancy and nursing, and
continued after weaning directly in the tested progeny as usual.
Tumor Scoring and Tissue Sampling.
After the specified duration of treatment, animals were killed by
CO2 inhalation. At the time of death, blood was
collected in heparinized tubes, and plasma was immediately separated
and frozen at -70°C for later a assay of drug levels and thromboxane
B2. Colon and small intestine were removed in
their entirety, opened longitudinally with fine scissors, and rinsed in
saline. Mucosal specimens were quickly obtained for ODC enzyme and
polyamine assays; colon and segments of small intestine (each 4.0 cm in
length) from the proximal (duodenum), middle (jejunum), and distal
(ileum) small intestine were then spread on individually labeled strips
of bibulous paper. They were fixed flat in 10% buffered formalin for
2 h, washed twice with 70% ethanol overnight, and then stained
with 0.2% methylene blue in Kreb’s Ringer solution for approximately
30 min. Tumors were counted by an individual, unaware of the animal’s
drug treatment status, using an Olympus SZH10 stereo dissecting
microscope to record tumor number, location, and diameter to a
precision of less than 0.1 mm.
Thromboxane B2 Assay.
Blood samples were collected in chilled polypropylene test tubes coated
with a solution of 4.5 mM EDTA and a prostaglandin
synthetase inhibitor (10 µg/ml indomethacin). Thromboxane
B2 in plasma was measured for each mouse using a
RIA kit (New England Nuclear Research Products, Boston MA) as described
previously (6)
. This rapid and sensitive RIA method
involves separation of antibody-antigen complexes from free antigen by
precipitation of antibody-bound tracer with polyethylene glycol in the
presence of carrier immunoglobulin. After centrifugation, the
supernatant containing the unbound antigen was decanted and was counted
in a gamma counter. The results obtained for the standards were used to
construct a dose-response standard curve from which the unknowns were
read by interpolation.
DFMO Assay.
DFMO in plasma was assayed by HPLC, using a procedure similar to that
of Smithers (30)
. Serum was collected at the time of death
from each of the animals in the DFMO-treated groups and the control
group and stored frozen until the time of assay. Plasma samples (100
µl) were extracted with 4 volumes of methanol after addition of the
internal standard, 4-amino-3-hydroxy butyric acid. The extracts were
derivatized with o-phthalaldehyde (OPA) and chromatographic
separation of the OPA-derivatized samples was achieved using a Waters
Nova-Pak cartridge, gradient elution with a methanol/pH 7.5 phosphate
buffer solvent system, and fluorescent detection (335 nm exitation, 450
nm emission). Quantitation compares the peak height of the DFMO samples
to that of the internal standard,4-amino-3-hydroxy butyric acid.
Piroxicam Assay.
Piroxicam was assayed in plasma by HPLC using a method based on that of
Macek and Vacha (31)
. Plasma standards or samples (100
µl of mouse plasma) were extracted with 0.3 ml of methanol after the
addition of the internal standard tenoxicam. The extract was diluted
1:1 with HPLC mobile phase and separation was achieved on a 15- cm
µBondapak CN column with an isocratic mobile phase of 30%
methanol/70% (pH 2.2) phosphate buffer. Detection was by UV absorption
at 360 nm. The amount of drug in samples is quantitated by comparison
with a standard curve of known piroxicam concentrations.
ODC Assay.
ODC activity in a soluble extract of colonic mucosa was determined by
measuring the release of CO2 from a radiolabeled
ornithine substrate (22)
. Intestinal mucosal samples
(total area, approximately 4 mm2) were
immediately transferred to vials containing 0.5 ml of an ice-cold
buffer [50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA,
and 0.1 mM pyridoxal phosphate)] and then homogenized and
centrifuged. The assay mixture contained 20 mM Tris-HCl (pH
7.5), 0.32 mM pyridoxal phosphate, 4 mM DTT,
0.4 mM EDTA, 12 µM L-ornithine
hydrochloride, 0. 02% Brij-35, containing 0.25 µCi
D,L-[1-14C]
ornithine hydrochloride and enzyme in a total volume of 0.25 ml. After
incubating at 37°C for 60 min in 15-ml Corex centrifuge tubes
equipped with rubber stoppers and center well assemblies, the reaction
was stopped by adding 0.5 ml of 2 M citric acid. The
incubation was continued for at least another hour to ensure complete
absorption of 14CO2 by the
ethanolamine:methoxyethanol (0.2 ml; 2:1 v/v) contained in the center
well. Finally, the center well containing the
ethanolamine:methoxyethanol was transferred to a vial containing 5 ml
of toluene-based scintillation fluid and 1 ml of ethanol, and the
associated radioactivity was determined in a liquid scintillation
counter.
Polyamine Assays.
The polyamines (spermine, spermidine and putrescine) were measured by
the HPLC assay method of Kabra and Lee (32)
.
Intestinal mucosal samples (approximately 3 mm2)
were obtained from each of the three segments of small bowel and from
areas of the colon not involved by tumors. These samples were
immediately transferred to vials containing 0.5 ml of 0.2 N
perchloric acid and stored in a freezer at -20°C until analysis.
Specimens were homogenized by a polytron in 0.2 N
perchloric acid and were then centrifuged. The supernatant was used for
polyamine analysis, and the pellet was used for the determination of
DNA content. Polyamine levels were expressed as pmol/µg DNA. After
the addition of the internal standard (1,7 diaminoheptane), the
polyamines in the acid extracts were derivatized with dansyl chloride,
and interferences were removed from the derivatized samples using
Bond-Elut C18 SPE columns. The derivatized polyamines were
separated on a Waters 8 x 10 Novapak C18 cartridge
using a gradient of 48–100% acetonitrile over 30 min against 10
mM sodium acetate buffer. Quantitation was accomplished
with fluorescent detection (excitation 340, emission 515). Polyamines
were quantitated by comparison of chromatographic peak areas of each
polyamine with that of the internal standard.
Statistical Design and Analysis.
The protocols used a one-way layout design for comparing the tumor
growth in Min mice under various chemopreventive strategies. The
randomization method that we used encourages balanced recruitment to
all of the treatment arms over time and diminishes the chance that
treatment effects would be confounded with potential time effects
(6
, 33)
. This method increases the likelihood that
experiments performed at different times will be comparable, making it
possible to compare the magnitude of drug effects in different studies.
Indeed, the tumor end points measured in the control groups in
different experiments in the Jacoby lab during the past 7 years have
been remarkably consistent. The tumor end points measured on all of the
animals were tumor multiplicity, tumor diameter, and the intestinal
location of each tumor along the longitudinal axis. All of the
measurements were reported as the mean ± SE.
In addition to controlling for unmeasured factors, the initial balanced randomization method enables an exact assessment of statistical significance after data collection. Specifically, a test statistic, such as a one-way F statistic, was calculated from the observed data and also from a large collection of hypothetical data sets, each one formed by reshuffling the observed data according to the randomization scheme. Exact Ps were computed by comparing the observed statistic with those simulated from this randomization distribution. Such analysis based on randomization makes accounting for multiple comparisons straightforward and minimizes the probability of calling any treatment significant when, in fact, no treatment differs from control. For comparison, we also calculated the traditional approximate normal theory Ps and accepted statistical significance only when both methods demonstrated P < 0.05.
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RESULTS |
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; and (b) we observed a significant dose-related inhibition
of plasma thromboxane levels (Fig. 1
) as expected for piroxicam because it is a nonspecific inhibitor of
both forms of COX (P = 0.051 for this
experiment overall).
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. Combined treatment with both DFMO and
piroxicam did not alter these plasma levels, which indicated there were
no significant drug interaction effects (Table 1)
. The biological
effect of DFMO in the target tissue was assessed by measuring levels of
the polyamine putrescine, which is the initial product of ODC activity.
Putrescine synthesis in the intestinal mucosa was decreased
significantly by DFMO treatment (Fig. 2
). There was no significant toxicity except for 9% decrease in
weight gain for the high-dose DFMO treatment group. DFMO inhibits ODC
activity (data not shown) and its initial product putrescine to
approximately one-half of control levels in both the colon and small
intestine (Fig. 2
), but the later products in the polyamine pathway
(spermidine and spermine) are not decreased to as great an extent (data
not shown).
|
. Adenomas in the distal small intestine had the most profound response
to treatment (Fig. 3
). In contrast, tumor multiplicity in the proximal portion of the small
intestine seems to increase moderately, and the limited number of
lesions that arise in the colon are minimally affected. Fig. 4
displays simultaneously the multiplicity (position of
circles) and average size (diameter of circles) for all
of the adenomas from each individual Min mouse in the proximal, middle,
and distal portions of the small intestine. Piroxicam clearly decreases
adenoma multiplicity; furthermore, the average size of the individual
lesions is markedly smaller throughout the intestine. Piroxicam at all
doses including the lowest dose of only 12 ppm has an effect, with the
magnitude of reduction becoming quite striking when using
doses 
25 ppm. Interestingly, this profound effect on
tumor size extends even to the proximal small intestine (Fig. 4
)
despite the fact that one sees an increase in the numbers of lesions in
this region in mice treated with piroxicam.
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DFMO Decreases Adenoma Multiplicity in Min Mice.
When DFMO was administered to Min mice beginning at the time that they
were weaned, we observed a dose-dependent inhibition of tumor
multiplicity, particularly in the distal (Fig. 4
) and also the middle
segments of the small intestine (Table 2)
. Interestingly, however, at
DFMO doses (0.5–1.0%) that decreased tumor multiplicity to an extent
similar to lower doses of piroxicam, there was no effect on tumor size
(Fig. 4
; Table 3
).
|
. Furthermore, the lesions in the distal
region of the small intestine that remained after treatment were
profoundly smaller than the tumors observed in control mice (Fig. 4
;
Table 3
). Many mice in the combined-treatment groups had very few or no
tumors, whereas all of the mice in the control group had more than 15
tumors (range, 15 to 83; mean, 43.1 ± 3.6).
Drugs Inhibiting Adenomas Also Select against Embryos with Apc
Mutation.
Lethality at an early stage of development occurs in
ApcMin/Min embryos that completely lack normal
Apc function due to homozygous mutation (34
, 35)
.
Therefore, the Min mouse line is maintained by crossing a mouse
heterozygous for this mutation (ApcMin/+) with a
C57BL/6J mouse that is wild type for the Apc gene
(Apc+/+). This cross has produced more than
6000 live births, with the expected 1:1 ratio of heterozygous
Apc mutant mice to homozygous-wild type mice, as predicted
by Mendelian genetics.
Because tumors arise quite rapidly at an early age, we thought it might
be necessary to begin treatment during development to achieve profound
chemopreventive effects. Thus, our initial studies compared
chemopreventive treatments begun during nursing and in
utero, with those administered only after weaning. Treatment of
pregnant mice decreased the number delivering litters from 88%, in
controls, to 60% in dams treated with 1.0% DFMO and only 50% in
those treated with 100 ppm piroxicam. Furthermore, the ratios of
mutant:wild-type genotypes in the offspring treated with DFMO or
piroxicam were significantly decreased to 0.26 (22:64) and 0.28
(18:46), respectively, in contrast to the Mendelian expected ratio of
0.49 (412:425) from control litters delivered at the same time (Table 4)
.
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DISCUSSION |
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The recent development of COX-2 specific agents is a promising alternative that may not have the negative side effects primarily associated with the additional inhibition of COX-1 activity by nonselective agents such as NSAIDs (24) . At least two COX-2 selective agents have proven to be relatively effective in the colon tumor models (3 , 25 , 28) . Another alternative is to use a lower dose of an NSAID, such as piroxicam, in combination with some other effective agent with different side effects. The agent we used here was the highly selective ODC inhibitor DFMO (18) . The enzyme ODC catalyzes the conversion of ornithine to putrescine, the initial rate-limiting step in the synthesis of polyamines. ODC expression is increased in a wide variety of human cancers and preinvasive lesions and acts as an oncogene in experiments in which it is ectopically overexpressed (14) . Prior preventive studies in the azoxymethane carcinogen-induced rat colon cancer model had shown that DFMO is highly effective (20 , 37) . DFMO has also been shown to be effective in various skin tumor models (18 , 38) . We have demonstrated that DFMO was moderately effective against adenoma formation in the Min mouse model. Given previous data indicating that DFMO may inhibit cellular proliferation, we expected that adenomas developing in the presence of DFMO would be much smaller than adenomas from control mice. However, there was no significant difference in tumor size after DFMO treatment. Perhaps it should not be surprising that effects on tumor size could be separate from multiplicity, because some agents may affect the growth rate independently of the initiation or establishment of adenomas. The lack of observed effect on tumor size after treatment with the polyamine pathway inhibitor DFMO contrasts sharply with the effects of piroxicam, a prostaglandin pathway inhibitor. Results with piroxicam alone were consistent with those of our previous study with that drug as a single agent, which indicated a decrease in both tumor multiplicity and size (6) . The few residual adenomas remaining after treatment with piroxicam are much flatter (decreased height above the mucosal plane) and about one-half the diameter compared with adenomas from untreated control mice. Piroxicam seems to cause adenoma regression similar to effects, caused by the specific COX-2 inhibitor celecoxib, that we previously reported.
The combination of relatively low doses of piroxicam together with DFMO profoundly decreased intestinal adenomas and was more effective than either drug alone. The appropriate intermediate markers (ODC enzyme activity and polyamine levels in the intestinal tissue or thromboxane B2 in blood) were decreased by DFMO and piroxicam in parallel to their tumor inhibitory effects. Tumor multiplicity and diameter were decreased significantly, with additive effects of this drug combination on both measures of the tumor phenotype. Despite the large number of tumors in every control animal, combination therapy almost completely suppressed adenomas in many of the treated mice. Although tremendous clinical efficacy would be necessary to prevent all adenomas in patients with severe polyposis phenotypes, combination treatments such as these used in conjunction with colonoscopic polypectomy might delay or obviate the need for colectomy in some patients. After patients have total colectomy, the next concern is periampullary tumors in the duodenum. In patients with subtotal colectomy, the rectum remains at high risk, and ileal pouch procedures may have some risk of adenomas. Our studies reveal that efficacy varies greatly depending on the region of intestine: proximal-mid-distal (duodenum-jejunum-ileum). Piroxicam is much more effective in the distal small intestine (ileum). The cause for this gradient of response is unknown but could be due either to luminal substances such as biliary secretions or growth factors or to intrinsic differences in gene expression in each intestinal region. Because duodenal tumors are clinically a very important problem in humans with familial adenomatous polyposis, our results with the Min mouse model suggest that careful studies of efficacy in the proximal small intestine are needed to assess the usefulness of NSAIDs in the familial adenomatous polyposis patient population.
At the very beginning of these experiments, the authors thought that it might be necessary to treat mice starting at a very early age, when adenomas begin developing, to achieve striking efficacy. The known major genetic change associated with adenoma formation—loss of the chromosome that bears the normal APC allele—may begin occurring while the animals are in utero. We, therefore, examined the effects of both DFMO and piroxicam in this model when initially administered to the dam. Routinely, after looking at thousands of offspring, one observes a genetic ratio of 1:1 (heterozygous APC mutant mice:homozygous APC wild-type mice) in crosses between heterozygous APC mutant mice and homozygous APC wild-type mice. In contrast, in similar crosses in which the dams were treated with piroxicam or DFMO, the resulting ratios were approximately 0.3:1 (heterozygous APC mutant mice:homozygous APC wild-type mice).
This ratio, together with the decreased litter size, implies that more than 60% of mice with an ApcMin/+ genotype were lost during development and that both of these agents, when administered in utero, were preferentially embryotoxic to mice with the mutant APC allele. Because the APC gene has not been previously shown to be directly associated with response to any chemical agents during development, the cause of this striking effect is still unknown.
In summary, these experiments demonstrate for the first time in a mouse model of adenomatous polyposis the profound efficacy of a combination-treatment approach, using relatively low doses of drugs that inhibit ODC and COX-2. Combined treatment of mice with piroxicam plus DFMO was much more effective than either agent alone and resulted in a significant number of mice totally free of any intestinal adenomas, in contrast to the 100% incidence and high multiplicity in control Min mice. In addition to this profound effectiveness in reducing tumor number, the few residual tumors in mice treated with the combined drugs were markedly smaller in size than tumors that arose from control Min mice. These experiments suggest that selective COX-2 inhibition combined with ODC inhibition is a very promising approach for colon cancer prevention.
ACKNOWLEDGMENTS
We appreciate the expert technical assistance of Marcia Pomplun.
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FOOTNOTES |
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1 Supported by National Cancer Institute Contract
N01 CN 65122 (awarded to R.F.J.). 
2 To whom requests for reprints should be
addressed, at H6/516 Clinical Science Center (5124), 600 Highland
Avenue, Madison, WI 53792. Phone: (608) 256-1901; Fax: (608)
280-7292.
3 The abbreviations used are: NSAID, nonsteroidal
anti-inflammatory drug; ODC, ornithine decarboxylase; DFMO,
difluoromethylornithine; GI, gastrointestinal; HPLC, high-performance
liquid chromatography; COX, cyclooxygenase.
Received 9/16/99. Accepted 2/ 3/00.
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A. UMAR, J. L. VINER, and E. T. HAWK The Future of Colon Cancer Prevention Ann. N.Y. Acad. Sci., December 1, 2001; 952(1): 88 - 108. [Abstract] [Full Text] [PDF]
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M. KATDARE, L. KOPELOVICH, and N. TELANG Chemopreventive Agents Inhibit Aberrant Proliferation of the Aneuploid Phenotype in a Colon Epithelial Cell Line Established from Apc 1638N [+/-] Mouse Ann. N.Y. Acad. Sci., December 1, 2001; 952(1): 169 - 174. [Abstract] [Full Text] [PDF]
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O. J. Sansom, L. A. Stark, M. G. Dunlop, and A. R. Clarke Suppression of Intestinal and Mammary Neoplasia by Lifetime Administration of Aspirin in ApcMin/+ and ApcMin/+, Msh2-/- Mice Cancer Res., October 1, 2001; 61(19): 7060 - 7064. [Abstract] [Full Text] [PDF]
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R. N. DuBois New paradigms for cancer prevention Carcinogenesis, May 1, 2001; 22(5): 691 - 692. [Full Text] [PDF]
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R. N. DuBois Cyclooxygenease-2 and Hepatocellular Carcinoma: Is It a Target for Prevention? Clin. Cancer Res., May 1, 2001; 7(5): 1110 - 1110. [Full Text]
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