Prevention by aspirin and its combination with
-difluoromethylornithine of azoxymethane-induced tumors, aberrant crypt foci and prostaglandin E2 levels in rat colon
Hong Li,
Herman A.J. Schut,
Philip Conran,
Paula M. Kramer,
Ronald A. Lubet1,
Vernon E. Steele1,
Ernest E. Hawk1,
Gary J. Kelloff1 and
Michael A. Pereira2
Medical College of Ohio, Department of Pathology, 3000 Arlington Avenue, Toledo, OH 43614-5806 and
1 Chemoprevention Branch, Division of Cancer Prevention and Control, National Cancer Institute, Rockville, MD 20892, USA
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Abstract
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The doseresponse relationship in male F344 rats was determined for the ability of aspirin administered in the diet to prevent azoxymethane (AOM)-induced colon cancer and aberrant crypt foci (ACF) and to reduce prostaglandin E2 (PGE2) levels. Starting at either 7 or 22 weeks of age, the rats received aspirin. All rats received two doses of AOM (15 mg/kg each on days 7 and 14) and were killed on day 36. The lowest concentrations of aspirin to prevent ACF or reduce PGE2 levels were 600 and 400 mg/kg, respectively. To evaluate the prevention of tumors, rats received either 0 or 400 mg/kg aspirin for a total of 39 weeks with AOM (30 mg/kg) administered 7 days after the start of treatment. Aspirin had no effect on the yield of colon tumors. In a second experiment, rats started to receive 0, 200, 600 or 1800 mg/kg aspirin or 1000 mg/kg
-difluoromethylornithine (DFMO) +/ aspirin. Eight and 15 days later, all the rats received 15 mg/kg AOM. Eleven weeks later, animals that were receiving the control diet started to receive 0, 200, 600 or 1800 mg/kg aspirin; 1000 or 3000 mg/kg DFMO; or 1000 mg/kg DFMO + 200 or 600 mg/kg aspirin. The animals were killed 32 weeks later. DFMO effectively reduced the yield of colon tumors when administered starting either before or after AOM while aspirin was much weaker. The combination of aspirin + DFMO administered after AOM was synergistic. Both aspirin and DFMO decreased the Mitotic Index, while apoptosis was increased only by DFMO. Our results demonstrated that aspirin and DFMO could prevent colon cancer when administered after AOM. Furthermore, aspirin reduced ACF, PGE2 levels and mitosis at concentrations that did not prevent cancer. In contrast, the ability to enhance apoptosis did correlate with the prevention of cancer.
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; COX, cyclooxygenase; DFMO,
-difluoromethylornithine; EHRT, Environmental Health Research and Testing Inc; NSAID, non-steroidal anti-inflammatory drug; ODC, ornithine decarboxylase; PGE2, prostaglandin E2.
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Introduction
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Colorectal cancer is the fourth most common type of cancer in the world (1). One strategy to decrease its incidence is the development of chemopreventive drugs. Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), including piroxicam and sulindac appear to be promising chemopreventive drugs for colon cancer (2). Although several epidemiological studies have suggested an inverse association between the chronic use of aspirin and the risk of colorectal cancer (36), one relatively large prospective study failed to observe an association (7). Aspirin has been shown to inhibit azoxymethane (AOM)-induced colon cancer in rats when provided in the diet before administering AOM and continuing until the end of the study (8). Wargovich et al. have reported that aspirin prevented aberrant crypt foci (ACF) when administered starting 4 weeks after AOM (9). However, similar concentrations of aspirin (200 and 400 mg/kg diet) administered starting prior to AOM did not prevent ACF (10). In the 1,2-dimethylhydrazine model of colon cancer, aspirin was effective in preventing colon tumors when administered during the initiation phase; however, it was not effective when administered 4 weeks after the carcinogen (11). Aspirin was also ineffective in preventing cholic acid promotion of AOM-initiated colon cancer (12). Thus, except for the prevention of AOM-induced ACF, aspirin has not been effective when administered only during the promotion/progression phase of carcinogenesis. In contrast two other NSAIDs, piroxicam and sulindac, have been very effective in preventing colon cancer when administered up to 14 weeks after AOM (1317).
NSAIDs inhibit cyclooxygenase (COX) and thus reduce the biosynthesis of prostaglandins. Aspirin preferentially acetylates COX-1 resulting in irreversible inhibition (2,18,19). Human colon tumors contain high levels of prostaglandins, in particular those of the E series (2023). High levels of prostaglandin E2 (PGE2) have also been reported in AOM-induced colon tumors (15). Therefore it is reasonable that inhibition of prostaglandin synthesis by NSAIDs might be associated with their prevention of colon cancer.
Ornithine decarboxylase (ODC) inhibitors are another class of chemicals that have shown to have efficacy in preventing cancer. ODC is the first enzyme in polyamine biosynthesis and is closely correlated with cell proliferation, differentiation and carcinogenesis (2426).
-Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has been shown to inhibit the development of cancer in several animal models (27) including prevention of AOM-induced colon cancer (16,17,2832). The inhibition by DFMO was dose-dependent and its combination with piroxicam was reported to be synergistic (16). DFMO was also effective in preventing AOM-induced ACF in rat colon (33).
While aspirin and DFMO are promising chemopreventive agents for colon cancer, there is limited understanding of the doseresponse relationship of aspirin and of their efficacy when administered in combination or only during the promotional/progression phase of carcinogenesis. Therefore, we evaluated the doseresponse relationship for the prevention by aspirin of AOM-induced ACF and colon tumors and for its reduction of PGE2 level in the colon. The ability to prevent colon tumors was also determined for aspirin and DFMO administered starting 11 weeks after AOM and for combinations containing them. Furthermore, the ability of aspirin, DFMO and the combination of them to alter the level of apoptosis and cell proliferation in colon adenomas is reported.
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Materials and methods
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Animals, chemicals and diet
Male F344 rats were obtained from Charles River Laboratories (Raleigh, NC). They were housed at the Medical College of Ohio (Toledo, OH), except for the first study for the prevention of colon tumor (experiment 3) that was housed at Environmental Health Research and Testing, Inc. (EHRT; Lexington, KY). Both Laboratory Animal Facilities are AAALAC accredited. The care and maintenance of the animals at both facilities were in accordance to the US Public Health Service `Guide for the Care and Use of Laboratory Animals'. Solid-bottom polycarbonate cages with stainless steel wire-bar lids and Bed-o-Cob bedding (Andersons, Toledo, OH) were used to house 2 rats/cage. The light cycle consisted of 12 h each of light and dark. The animal rooms were maintained at 6476°F and 55 ± 15% relative humidity. Drinking water and AIN 76A diet were supplied to the animals ad libitum.
AOM was purchased from Sigma (St Louis, MO) and aspirin was from Acros Organics (Pittsburgh, PA), except in the study performed at EHRT where it was purchased from ICN Biochemicals (Irvine, CA). DFMO was obtained from the DCPC Repository of the NCI (c/o McKesson BioServices, Rockville, MD). AIN-76 diet was purchased from Dyets (Bethlehem, PA) and used within 3 months of formulation.
Assays for dose selection, prevention of ACF and reduction of PGE2 levels
Toxicity, prevention of AOM-induced ACF and reduction of PGE2 levels were evaluated in two experiments in which aspirin was administered in the diet of rats starting either at 7 (experiment 1) or 22 (experiment 2) weeks of age. These two ages corresponded to when the rats started to receive aspirin in the second study for prevention of colon cancer. In experiment 1, rats at 7 weeks of age were assigned to one of six treatment groups consisting of 10 animals each and received either 0, 200, 400, 600, 1000 or 2000 mg aspirin/kg diet. In experiment 2, the rats were 22 weeks of age when assigned to eight treatment groups of 11 animals each and started to receive either 0, 200, 400, 600, 1000, 2000, 3000 or 4000 mg/kg aspirin in their diet. In both experiments at 7 and 14 days after the rats started to receive the aspirin, they were administered AOM (15 mg/kg) by i.p. injection. The animals received aspirin for a total of 5 weeks (3 weeks after the second dose of AOM) and were then killed by carbon dioxide asphyxiation. Body weight was monitored at assignment to the treatment groups so that there was no difference among the groups and weekly during exposure to aspirin.
At necropsy, the colons were removed, cut along the longitudinal axis and flushed with cold saline. For determination of PGE2, a sample of the mucosa was obtained by scraping the 04 cm segment of colon from the anus. Using liquid nitrogen, it was rapidly frozen in vials coated with 10 µg/ml indomethacin and stored at 70°C. The rest of the colon was then stained and evaluated for ACF as described by Bird (34) and previously used by us (10,33). Briefly, they were stained for 10 min in a solution of 0.2% methylene blue (Sigma) dissolved in 70% alcohol. After staining, the colons (mucosal side up) were placed on a microscope slide and with the aid of a light microscope (magnifications of 40 and 100x) evaluated for ACF. The criteria used to identify ACF included the following: (i) increased size of the crypts; (ii) increased thickness of the epithelial cell lining; (iii) elongation of the luminal opening; and (iv) increased pericryptal zone separating the crypts of the ACF from the surrounding normal appearing crypts. The location of the ACF in the colon and the number of crypts/focus were recorded.
Determination of the concentration of PGE2
A sample of the mucosal scraping was sonicated for 10 s in 1.0 ml 0.1 M TrisHCl buffer, pH 7.4, containing 0.02 M EDTA and 10 µg/ml indomethacin. A 0.2 ml aliquot was removed for determination of protein content using the Bio-Rad DC Protein Assay kit (Richmond, CA). To the remainder of the sample, 0.8 ml of methanol was added and vigorously mixed. After acidifying the sample with six drops of 1% formic acid, it was extracted with 1.5 ml chloroform. Following centrifugation at 2000 r.p.m. for 10 min, the organic phase was removed and dried using a Speed-Vac. The dried extract was resuspended in assay buffer and assayed for PGE2 content using Dupont125 I-PGE2 RIA kit (Boston, MA). PGE2 concentration was calculated as pg PGE2/mg protein.
Assays for prevention of colon tumors
In experiment 3, six-week-old male F344 rats were divided into the following treatment groups with no significant difference in body weight among them. Groups 1 and 2 contained 30 and 10 animals, respectively, and received 400 mg aspirin/kg diet. Groups 3 and 4 contained 30 and 10 animals, respectively, and continued to receive the control AIN 76A diet. Seven days later the animals in treatment groups 1 and 3 received an s.c. injection of AOM (30 mg/kg body wt) and those in groups 2 and 4 received 4.0 ml/kg of the saline vehicle. The animals continued to receive the assigned diets until killed by carbon dioxide asphyxiation 38 weeks after receiving the AOM. Body weight was monitored weekly for the first 4 weeks and every 23 months thereafter.
Experiment 4: in the above two dose-selection studies, aspirin concentrations of 2000 (experiment 1) and 4000 (experiment 2) mg/kg diet did not appear to affect the health and body weight of the animals. Furthermore, the reduction by aspirin of the yield of ACF and of the level of PGE2 reached maximums by 6002000 mg/kg diet as discussed later in Results. Therefore, in this experiment 1800 mg/kg diet was chosen as the high concentration of aspirin. Male F344 rats (7 weeks old) were divided into 14 treatment groups containing 34 animals each so that there was no significant difference in the body weights among the groups. Then the animals in treatment groups 16 started to receive in their AIN 76A diet either 0 (control diet), 200, 600 or 1800 mg/kg aspirin or 1000 mg/kg DFMO + 0, 200 or 600 mg/kg aspirin. Eight and 15 days later, all the rats in the 14 treatment groups were administered AOM (15 mg/kg body wt) by i.p. injection. Treatment groups 714 continued to receive the AIN-76A diet until 11 weeks after the second dose of AOM. At that time, the animals in groups 713 received in their diet either 200, 600 or 1800 mg/kg aspirin; 1000 or 3000 mg/kg DFMO; or 1000 mg/kg DFMO + 200 or 600 mg/kg aspirin. Treatment group 14 continued to receive the AIN-76A diet. The animals were fed their respective diets until they were killed 43 weeks after the second dose of AOM. Body weight was monitored weekly for the first 6 weeks and monthly thereafter throughout the experiment.
At necropsy in experiments 3 and 4, the colons were removed from cecum to anus, slit open longitudinally, flushed with saline and examined for tumors. The location and size of the tumors were recorded prior to harvesting. The small intestine was palpated for the presence of tumors that were also harvested. The tumors were fixed in 10% buffered formalin overnight and then stored in 70% alcohol until embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin for histopathologic evaluation.
Determination of apoptosis and cell proliferation in adenomas
Adenomas from treatment groups in experiment 4 that received 0, 600 or 1800 mg/kg aspirin; 1000 mg/kg DFMO; or 1000 mg/kg DFMO + 600 mg/kg aspirin were evaluated for apoptosis and mitosis. Only adenomas were evaluated because of the limited number of carcinomas. Apoptotic and mitotic cells were counted using H & E stained sections. Apoptotic cells were identified by cell shrinkage with a halo separating them from surrounding cells, nuclear condensation, formation of apoptotic bodies (nuclear fragments), and eosinophilic and condensed cytoplasm (35). At least 1000 epithelial cells were evaluated and the number of apoptotic and mitotic cells recorded. The Apoptotic and Mitotic Indexes were determined by dividing the number of affected cells by the total number of cells evaluated multiplied by 100.
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Results
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Experiments 1 and 2: dose selection, prevention of ACF and reduction of PGE2
In experiments 1 and 2, aspirin administered for 5 weeks at concentrations as high as 2000 and 4000 mg/kg diet did not affect the body weight of the animals or their food consumption. There was also no indication of toxicity observed during the daily evaluation of the animals. Therefore, the maximum concentrations of aspirin evaluated in experiments 1 and 2, i.e. 2000 and 4000 mg/kg diet, would appear not to be toxic.
The effect of aspirin administered to rats starting at 7 or 22 weeks of age on AOM-induced ACF and on PGE2 level in the colon is presented in Figure 1
. Aspirin caused a dose-dependent reduction in the yield of ACF and in the level of PGE2. The lowest concentration of aspirin in both experiments to significantly prevent ACF was 600 mg/kg and to reduce PGE2 was 400 mg/kg. In the two experiments, maximum reduction of ACF was 4045% (6002000 mg/kg), while for PGE2 levels was 6585% (10002000 mg/kg). Hence, although both prevention of ACF and reduction of PGE2 reached maximums at similar concentrations of aspirin, the reduction of PGE2 was greater than the prevention of ACF. The yield of ACF of all sizes were reduced, i.e. foci with 1, 2 or 3+ crypts/focus.


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Fig. 1. Inhibition by aspirin of ACF and PGE2 in rats of 7 (A) and 22 (B) weeks of age at the start of the experiments. Results are the percentage inhibition when compared with the AOM + control diet group and are means ± SE for treatment groups consisting of 10 (A) and 11 (B) animals each. The yield of ACF and the level of PGE2 were 98.0 ± 10.9 ACF/animal and 93.2 ± 8.7 pg PGE2/mg protein and 159.0 ± 10.0 ACF/animal and 285.2 ± 54.8 pg PGE2/mg protein in the AOM + control diet group for 7- and 22-week-old rats, respectively. *Significant difference from the AOM + control diet group by one way ANOVA followed by the Tukey test, P < 0.05.
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Experiments 3 and 4: prevention of colon tumors
In experiment 3, the prevention of colon cancer was examined for 400 mg/kg of aspirin administered in the diet from 1 week before AOM until killed 38 weeks later. Aspirin did not affect the body weight of the animals during the experiment including the terminal killing presented in Table I
. Table I
also contains the yield of colon adenomas, adenocarcinomas and tumors (adenomas + adenocarcinomas). Aspirin did not affect the yield or incidence of animals with these neoplastic lesions. No tumors were found in animals that did not receive AOM.
In experiment 4, aspirin, DFMO and combinations containing them were administered starting either before or 11 weeks after AOM (Table II
). Aspirin, DFMO or their combinations did not affect the body weight of the animals during the study. Table II
contains the body weight of the animals at killing and the incidence of animals with colon adenomas, adenocarcinomas or tumors (adenomas + adenocarcinomas). The multiplicity of adenomas, adenocarcinomas and tumors/animal are presented in Figures 2 and 3
. When administered starting before AOM, concentrations of aspirin up to 1800 mg/kg diet did not significantly alter the incidence (Table II
) or multiplicity (Figure 2
) of adenomas, adenocarcinomas or tumors. When administered starting 11 weeks after AOM, the high concentration of aspirin (1800 mg/kg diet) but not its two lower concentrations, significantly reduced the multiplicity of adenomas and tumors (Figure 3
). However, aspirin did not effect the incidence of animals with adenomas, adenocarcinomas or tumors (Table II
).

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Fig. 2. Effect of aspirin and DFMO administered starting before AOM on the multiplicity of neoplastic lesions in the colon. Results are means ± SE. The data were statistically analyzed by a one way ANOVA followed by the Tukey test. The * indicates P < 0.05 when compared with the AOM + control diet group; a, P < 0.05 when the combinations were compared with the treatment group that received only DFMO.
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Fig. 3. Effect of aspirin and DFMO administered after AOM on the multiplicity of neoplastic lesions in the colon. Results are means ± SE. The data were statistically analyzed by a one way ANOVA followed by the Tukey test. *P < 0.05 when compared with the AOM + control diet group. None of the combinations was statistically different from the treatment group that received only DFMO.
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DFMO administered starting before AOM reduced the multiplicity of adenomas and tumors (Figure 2
) and the incidence of animals with adenomas although the reduction in the incidence was not statistically significant (P = 0.069) (Table II
). When DFMO was administered in combination with either 200 or 600 mg/kg aspirin, the incidence and multiplicity of adenomas, adenocarcinomas and tumors were reduced further with a dose-dependent trend. When administered after AOM, DFMO (1000 and 3000 mg/kg diet) reduced both the incidence and multiplicity of adenomas and tumors (Table II
, Figure 3
). The combination of either 200 or 600 mg/kg aspirin with DFMO (1000 mg/kg diet) administered after AOM did not reduce further the incidence or multiplicity of adenomas, adenocarcinomas or tumors.
Effect of aspirin and DFMO on apoptosis and mitosis in colon tumors
The effect of 600 and 1800 mg/kg aspirin, 1000 mg/kg DFMO and the combination of 1000 mg/kg DFMO + 600 mg/kg aspirin on the Apoptotic and Mitotic Indexes in adenomas is presented in Table III
. Aspirin administered at either 600 or 1800 mg/kg, DFMO and the combination of DFMO + aspirin reduced the Mitotic Index in adenomas by 4963%. DFMO with and without 600 mg/kg aspirin increased the Apoptotic Index 2.79- and 3.70-fold, respectively. However, aspirin (600 and 1800 mg/kg diet) administered by itself did not affect the Apoptotic Index.
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Discussion
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Epidemiological and experimental studies have suggested a low to moderate efficacy of aspirin to prevent colon cancer (29,11,12,36). Reddy et al. reported that 200 and 400 mg/kg aspirin in the diet administered starting before AOM and continuing for 52 weeks significantly reduced the incidence and multiplicity of colon tumors in male F344 rats (8). However, we were unsuccessful in confirming prevention by 400 mg/kg aspirin. Others have found that aspirin at greater concentrations in the diet including 4000 mg/kg were not toxic to rats (3739). Therefore, we first confirmed that aspirin concentrations of 2000 and 4000 mg/kg diet were not toxic and then determined the effect of non-toxic concentration >400 mg/kg (600 and 1800 mg/kg) on the yield of AOM-induced colon cancer. When administered starting before and/or after AOM, aspirin at 200, 600 and 1800 mg/kg diet did not affect the yield of colon tumors except for a reduction resulting from the highest concentration of aspirin administered after AOM. Aspirin administered to rats at 1800 mg/kg diet is equivalent to ~240 mg/kg body wt, 16.8 g/70 kg person or 3.36 tablets (5 g tablets)/day. The low activity of this relatively high daily dose of aspirin could account for the conflicting results of several epidemiological studies that found an inverse association between chronic use and the risk of colorectal cancer (36), while one relatively large prospective study failed to observe an association (7).
Compared with other NSAIDs, the prevention of colon cancer by aspirin is relatively weak. Piroxicam and sulindac have been reported to prevent colon cancer at concentration as low as 25 and 160 mg/kg diet (13,15). Aspirin is primary a COX-1 inhibitor, while these two NSIADs inhibit both COX-1 and 2 (2,18,49). The level of COX-2 in the colon has been associated with the development of tumors. Hence, the greater ability of the other two NSAIDs to inhibit COX-2 could explain their greater efficacy in preventing AOM-induced colon cancer (1317).
DFMO has previously been reported to prevent colon cancer (17,30) and ACF (33) when administered starting before AOM. The present study demonstrated that DFMO was also effective in preventing colon tumors when administered starting 11 weeks after AOM (Table II
, Figure 3
). There was no significant difference between the efficacy of DFMO administered before and/or after AOM. Aspirin only significantly prevented colon tumors when administered after AOM. Thus much of the ability of aspirin and DFMO to prevent colon cancer would appear to occur during the promotion/progression phase.
The combination of DFMO with aspirin appeared to be more effective in preventing colon tumors than DFMO. Although, aspirin at 200 and 600 mg/kg did not prevent colon cancer, when administered with DFMO starting before AOM they caused an apparent dose-related reduction of colon tumors (Figure 3
). The combination of DFMO with piroxicam has been reported to be synergistic in preventing colon cancer when administered before AOM (16,17). When administered after AOM, we did not observe an enhancement of the efficacy of DFMO by the addition of aspirin (200 and 600 mg/kg). The inability to detect an additive or synergistic effect of aspirin was probably due to the already low yield of colon tumors in the presence of DFMO (<0.5 tumors/rat). Hence, evaluation of lower concentrations of DFMO that cause less of a reduction in tumor yield appears to be required to demonstrate synergism with aspirin.
In the two studies to examine the toxicity of aspirin, we also determined its effect on the yield of AOM-induced ACF and on PGE2 levels in the colon. Aspirin concentrations of as low as 600 and 400 mg/kg diet produced significant reduction in the yield of ACF and the level of PGE2, respectively. Wargovich et al. (9) have reported prevention of ACF by 200 and 400 mg/kg aspirin administered 4 weeks after AOM. We achieved maximum reduction of the yield of ACF and of the level of PGE2 with 6002000 and 10002000 mg/kg diet, respectively. Hence, concentrations of aspirin that resulted in maximum prevention of ACF and reduction of PGE2 levels did not prevent colon cancer. Since, aspirin is primary a COX-1 inhibitor, we propose that it is more active in preventing ACF and reducing PGE2 levels because this is the primary isoform in the mucosa, while tumors because of their COX-2 activity are less sensitive.
Both increased apoptosis and/or decreased cell proliferation have been proposed as mechanisms for the chemopreventive activity of NSAIDs and other agents (4045). However, the enhancement of apoptosis rather than the reduction of cell proliferation has been more consistently associated with the ability of NSAIDs to inhibit human cancer cells in culture (40,41,43,44) and of sulindac to induce the regression of polyps in FAP patients (46,47). Samaha et al. reported that other chemopreventive agents including two NSAIDs (curcumin and sulindac) and phenylethyl-3-methylcaffeate enhanced the Apoptotic Index in AOM-induced colon tumors (48). In our study, DFMO and the combination of DFMO + aspirin decreased the Mitotic Index and enhanced apoptosis in adenomas and prevented colon cancer. However, both 600 and 1800 mg/kg aspirin were very effective in decreasing the Mitotic Index while not affecting apoptosis and very weakly preventing colon cancer by only the high concentration. Similarly, we have found that the prevention of AOM-induced colon cancer by piroxicam (unpublished data) and retinoids was more closely associated with enhancement of apoptosis than with the reduction of cell proliferation and the prevention of ACF (49). However, the prevention by retinoids of AOM-induced ACF was highly associated with decreased cell proliferation (50). Hence, the enhancement of apoptosis appears to be associated with prevention of colon cancer while decreased cell proliferation is more associated with prevention of ACF. This would suggest that the screening of agents for prevention of colon cancer should include prevention of ACF followed by an evaluation for the ability to enhance apoptosis in order to distinguish agents that prevent ACF but do not prevent colon cancer.
In summary, we report that aspirin and DFMO were effective in preventing colon cancer when administered 11 weeks after AOM during the promotional/progression phase of carcinogenesis. However, aspirin had only very weak activity at the relatively high concentration of 1800 mg/kg diet. Nevertheless aspirin at much lower concentrations did demonstrate dose-related prevention of ACF and reduction of PGE2 levels in the colon. The combination of aspirin with DFMO when administered before AOM was synergistic in preventing colon cancer. The strong chemopreventive activity of DFMO and the weak activity of aspirin were more closely associated with enhancement of apoptosis than with decreased cell proliferation.
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Acknowledgments
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This work was supported in part by the National Cancer Institute, contracts NO1-CN-2529501, NO1-CN-5517401 and NO1-CN-5517501.
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Notes
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2 To whom correspondence should be addressed Email: mpereira{at}mco.edu 
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Received July 13, 1998;
revised October 14, 1998;
accepted October 30, 1998.