Carcinogenesis vol.21 no.4 pp.617–621, 2000
Chemoprevention of familial adenomatous polyposis development in the APCmin mouse model by 1,4-phenylene bis(methylene)selenocyanate
Chinthalapally V.Rao2, Indranie Cooma, Jose G.Rosa Rodriguez1, Barbara Simi, Karam El-Bayoumy1 and Bandaru S.Reddy Chemoprevention Program, Division of Nutritional Carcinogenesis and 1Division of Cancer Etiology and Prevention, American Health Foundation, Valhalla, NY 10595, USA 2To
whom correspondence should addressed at: American Health Foundation, 1 Dana Road, Valhalla, NY 10595, USA Email:
[email protected]
Epidemiological and experimental studies have suggested that dietary supplementation with selenium can inhibit the development of cancers at several organ sites. We have consistently shown that 1,4-phenylene bis(methylene) selenocyanate (p-XSC) is a highly effective cancer chemopreventive agent against the development of chemically induced cancers in several laboratory animal species. This is the first report describing the preventive effects of p-XSC in an animal model of familial adenomatous polyposis (FAP) containing a germline mutation of the APC gene. Six-week old male (heterozygous) C57BL/6J-APCmin or wild-type mice were fed high fat diets containing 0, 10 or 20 p.p.m. p-XSC. After 80 days, the mice were killed and their intestines were excised and evaluated for polyps. Multiple samples were also harvested from normal appearing small intestine and colon for molecular analysis. Both the mucosa and polyps from the intestine and colon were assayed for β-catenin, cyclooxygenase (COX)-2 expression and COX isoform activities. Administration of p-XSC in the diet significantly decreased the rate of formation of small intestinal tumors (P < 0.0001) and colon tumors (P < 0.002) in APCmin mice. p-XSC produced a dose-dependent inhibition of tumors in both small intestine (P < 0.0001) and colon (P < 0.035). Mice fed 20 p.p.m. p-XSC had significantly lower levels of β-catenin expression and COX-2 activity in polyps. These observations demonstrate for the first time that the synthetic organoselenium compound p-XSC possesses antitumor activity against genetically predisposed neoplastic lesions, such as FAP. While the exact mechanism(s) for this antitumor activity of p-XSC remains to be elucidated, it appears that modulation of β-catenin expression and COX-2 activity is associated with inhibition of intestinal polyps. Introduction Colorectal cancer is one of the leading causes of cancer death in the Western world, including North America (1). Several epidemiological and laboratory studies suggest a relationship between the development of colon cancer and exogenous Abbreviations: AA, arachidonic acid; APC, adenomatous polyposis coli; COX, cyclooxygenase; FAP, familial adenomatous polyposis; HETE, hydroxyeicosatetraenoic acid; MTD, maximum tolerated dose; PG, prostaglandin; p-XSC, 1,4-phenylene bis(methylene)selenocyanate; SC, selenocysteine; SM, selenomethionine; Tcf, transcription factors. © Oxford University Press
factors, such as diet. Available evidence also suggests involvement of endogenous factors, such as individual genetic susceptibility. Thus, the etiology of colon cancer is multifactorial and complex (2,3). The progression from normal epithelium to colon cancer is a multistep process involving accumulation of multiple genetic alterations. Adenomatous polyposis coli (APC), a tumor suppressor gene on chromosome 5q, is considered to be a gatekeeper in colon tumorigenesis (4). Its inactivation leads to the development of adenomatous polyps (5,6). In most cases of colonic neoplasia, both inherited and somatic, the APC gene is either mutationally inactivated by the introduction of premature stop codons and/or deleted by loss of heterozygosity (6). Several mouse lineages that are heterozygous for specific mutations at the endogenous APC gene have been developed and characterized with respect to their intestinal multiple tumors (reviewed in ref. 7). Among mutant APC mice, APCmin, an inbred strain of C57BL/6J, carries a germline truncation of one APC allele (5): these mice develop multiple intestinal adenomas by 4 months of age. This model has been utilized to unravel the basic mechanisms of intestinal tumor formation and for testing cancer chemopreventive agents. Studies of the biochemical mechanisms downstream of APC mutations have provided important leads. Overexpression of cyclooxygenase (COX)-2 is one of the most significant observations in this respect (8). Oshima et al. (8) elegantly revealed in that study expression of COX-2 and its influence on the number of intestinal polyps and suppression of polyp formation by inhibitors of COX isoforms. This study provided definitive evidence that induction of COX-2 is an early, rate limiting step for adenoma formation. Recent studies also suggest another mechanism for APC function, namely that APC binds to β-catenin, which in turn binds to and regulates the transcription factor LEF-1 (4,9). Selenium, an essential trace element in humans, has received considerable attention for its possible role as a chemopreventive agent. Epidemiological studies have revealed an inverse association between dietary selenium intake and colon cancer risk in humans (10). A protective role for selenium was also observed in chemically induced carcinogenesis in laboratory animals (11,12). Human exposure to selenium occurs primarily via ingestion of grains and vegetables that contain organic forms of the element, such as selenomethionine (SM) and selenocysteine (SC). Cancer prevention studies (11) have not revealed any significant differences whether selenium was derived from inorganic or natural sources such as SM and SC. However, chronic feeding of either form of selenium at levels ⬎5 p.p.m. produced toxic effects (13). A study with Se-methylselenocysteine, a major constituent of selenized garlic, has shown some advantages as compared with selenomethionine (14). Therefore, substantial efforts have been made to develop and test organoselenium compounds that have maximal chemopreventive efficacy but lowest possible toxicity (11,15). During the past decade, studies conducted in our laboratories (11,15–20) have indicated that certain synthetic 617
C.V.Rao et al.
organoselenium compounds hold great promise as chemopreventive agents. These agents were found to be superior to historically used selenium compounds, such as sodium selenite, and to the naturally occurring SM and SC. Among the organoselenium compounds developed, 1,4-phenylene bis (methylene)selenocyanate (p-XSC) was far less toxic (LD50 values for l-selenomethionine, Na2SeNO3, benzyl selenocyanate and p-XSC in rats are 26, 39, 125 and ⬎1000 mg/kg body wt, respectively), yet more efficient in inhibiting development of cancers of the colon, mammary glands, lung and oral cavity (15–20). Our studies in the rat colon cancer model indicated that 80% of the maximum tolerated dose (MTD) of dl-selenomethionine (10 p.p.m.), Na2SeNO3 (4 p.p.m.) and benzyl selenocyanate (25 p.p.m.) produced 45, 16 and 50% inhibition of azoxymethane-induced colon carcinogenesis (15,18). Interestingly, administration of p-XSC at 30% of the MTD (20 p.p.m.) level inhibited ⬎60% of azoxymethaneinduced colon cancer in rats (21). Until now there were no studies to demonstrate the chemopreventive efficacy of p-XSC in APCmin mice, which develop multiple intestinal adenomas relevant to human familial adenomatous polyposis (FAP). Therefore, the present study was designed to evaluate the chemopreventive effects of two dose levels of dietary p-XSC on intestinal tumorigenesis in APCmin mice. To understand the underlying mechanism of action, we studied the modulatory effect of p-XSC on β-catenin expression and COX isoform expression and activities in intestinal polyps of this genetically well-defined intestinal tumor model. Materials and methods Animals, diets and p-XSC Heterozygotic male Min (C57BL/6J-APCMin/⫹Apc) and wild-type C57BL/6J male mice were obtained at 5 weeks of age from Jackson Laboratories (Bar Harbor, ME). All ingredients for the semi-purified diets were purchased from Dyets Inc. (Bethlehem, PA) and stored at 4°C prior to preparation of the diets. The composition of the high fat semi-purified diet was: casein, 21.3%; corn starch, 43.5%; dextrose, 8%; corn oil, 17%; alphacel, 5%; AIN mineral mix, 3.5%; AIN revised vitamin mix, 1.2%; d,l-methionine, 0.3%; choline bitartrate, 0.2%. The test agent (p-XSC) was blended with the diet by use of a V-blender (Patterson-Kelley Co., East Stroudsburg, PA) after it had first been premixed with a small quantity of diet in a food mixer. The control diet and experimental diets containing p-XSC were prepared weekly in our laboratory and stored in a cold room. The p-XSC content of the experimental diets was determined periodically (every 3 weeks) in multiple samples taken from the top, middle and bottom portions of individual diet preparations to verify uniform distribution (18). p-XSC was synthesized as described (17). Its purity was ⬎99%, as ascertained by high performance liquid chromatography analysis (17). The dose of p-XSC added to the diet was based on our earlier studies (18). In this study we used 10 and 20 p.p.m. p-XSC (5 and 10 p.p.m. as selenium). The control high fat diet contained ~0.1 p.p.m. selenium in the form of Na2SeO3. Experimental procedure Following a 1 day quarantine, all mice were distributed so that average body weights in each group were about equal (10 mice in each treatment group and six wild-type mice as parallel treatment groups). The animals were then transferred to a holding room, where they were housed individually in plastic cages with filter tops. Laboratory conditions were controlled to maintain a 12 h light/dark cycle at 50% relative humidity and at 21 ⫾ 1°C. The mice were fed the control or experimental diets until termination of the study, i.e. for ~80 days. The animals and their food cups were weighed thrice weekly and checked daily for signs of weight loss or lethargy that might indicate intestinal obstruction or anemia. At 120 days of age, all mice were killed by CO2 asphyxiation. This point in time was chosen to minimize the risk of intercurrent mortality caused by severe progressive anemia, rectal prolapse or intestinal obstruction, which usually occurs among Min mice at ~120 days of age (7). After necropsy, the intestinal tracts were dissected from the esophagus to the distal rectum, spread onto filter paper, opened longitudinally with fine scissors and cleaned with sterile saline. They were examined under a dissection microscope with 5⫻ magnification for tumor counts. This procedure was followed by two individuals who were blind to the experimental group and
618
the genetic status of the mice. Tumors that required further histopathological evaluation (masses that were not clearly identified, such as sessile tumors and enlarged lymph nodes) were fixed in 10% neutral-buffered formalin, embedded in paraffin blocks and processed by routine hematoxylin and eosin staining. In addition, multiple samples of tumors from the small intestines and colons were sectioned and stored in liquid nitrogen for analysis of COX activities and β-catenin levels. Western blot analysis of β-catenin and isoforms of COX Intestinal polyps isolated from individual mice were combined to obtain sufficient tissue (6–8 samples/group). Normal appearing intestinal mucosal samples were homogenized in 1:3 vol of 100 mM Tris–HCl buffer (pH 7.2) with 2 mM CaCl2. After centrifugation at 100 000 g for 1 h, the resulting separations were subjected to 8% SDS–PAGE for COX isoforms (particulate only) and β-catenin (both cytosol and particulate). Purified COX-1 and COX2 proteins (Cayman Chemicals, Ann Arbor, MI) and β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as electrophoresis standards. The proteins were electroplated onto PVDF nitrocellulose membranes as described previously (8). Goat polyclonal antibodies against COX-1, COX-2 and β-catenin were purchased from Santa Cruz Biotechnology. After blocking the membranes in 5% non-fat dry milk, they were incubated with anti-COX2 (1:2500) and anti-β-catenin (1:1500) antibodies for 1 h. The membranes were washed three times and incubated once more with secondary horseradish peroxidase-linked anti-goat IgG antibody (Santa Cruz Biotechnology) at a final concentration of 1:2000. The membranes were developed by the ECL chemiluminescence system (Amersham Life Science, Arlington Heights, IL) and exposed to Kodak XAR5 film. Intensities of each band were scanned by a computing densitometer. COX-1 and COX-2 activities Activities in intestinal tumor samples were assayed using a slight modification of a previously published method (22). Briefly, 150 µl of the reaction mixture containing 12 µM [14C]arachidonic acid (AA) (420 000 d.p.m.), 1 mM epinephrine, 1 mM glutathione in 50 mM phosphate buffer (pH 7.4) and 30 µg of microsomal protein from intestinal polyps were used for each assay. To determine COX-1 activity, the reaction mixtures were preincubated with 50 µM celecoxib, a selective inhibitor that blocks COX-2 isoform activity. For determination of COX-2 activity, the reaction mixtures were preincubated with 150 µM aspirin to block COX-1 activity and to modify COX-2 activity to 15-(R)-hydroxyeicosatetraenoic acid (HETE) (23,24). After incubation at 37°C for 20 min, the reaction was terminated by adding 40 µl of 0.2 M HCl. The COX-mediated metabolites of AA were extracted with ethylacetate (3⫻0.5 ml). The combined extracts were evaporated to dryness under N2, dissolved in chloroform and subjected to TLC on precoated plastic plates (silica G60, 20⫻20 cm, layer thickness 150 µm). The TLC plates were developed with a solvent system containing chloroform/methanol/acetic acid/ water (100:15:1.25:1 v/v/v/v). They were then exposed in an iodide chamber for 5 min to visualize the standards. The metabolites of [14C]AA, corresponding to prostaglandin (PG)E2, PGF2α, PGD2, 6-keto-PGF1α and thromboxane B2 for COX-1 activity and 15-(R)-HETE for COX-2 activity, were detected by their co-migration (Rf values) with authentic standards. The area of each metabolite was determined in a Bioscan System 200 image scanning counter (Bioscan, Washington, DC) equipped with a β-detector. Statistical analyses Differences in the body weights and tumor multiplicities (i.e. no. of tumors/ animal) were compared among the groups based on one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons procedure in which several treatment groups are compared with a control. COX-1, COX-2 and β-catenin expression was compared between the mice fed control diet and those given p-XSC-containing diets. All results are expressed as means ⫾ SEM and were analyzed by Student’s t-test. Differences were considered significant at the P < 0.05 level.
Results General observations Body weights of the APCmin mice that consumed p-XSC in their diets were in general somewhat higher and the mice were less anemic than those fed a control diet. However, no statistically significant (P ⬎ 0.05) differences in body weights were observed between the dietary groups (data not shown). As expected, APCmin mice began to lose body weight at ~90 days of age, due to intestinal obstruction and progressive anemia. In wild-type mice, the administration of p-XSC did
Prevention of polyps in APCmin mouse by p-XSC
Fig. 1. Effect of p-XSC on small intestinal tumor formation in APCmin mice. Detailed information is given in Materials and methods.
intestinal polyps and mucosa is summarized in Table I. According to absorbency measured by laser densitometric scans of western blots, mice that were fed 20 p.p.m. p-XSC had significantly reduced (⬎53%, P ⬍ 0.001) expression of membrane-bound β-catenin in the polyps but showed no significant effect (–9.6%, P ⬎ 0.05) on mucosal β-catenin levels. Also, administration of the p-XSC diet did not significantly affect cytosolic β-catenin levels in adenomatous polyps (data not shown). As expected, both COX-1 and COX2 protein levels were significantly higher in polyps than in adjacent mucosa. Administration of 20 p.p.m. p-XSC had no significant effect on expression of COX-1 (6.1%) and COX-2 (12.3%, P ⬎ 0.05) in intestinal polyps. Table II summarizes the observed modulatory effects of 20 p.p.m. p-XSC with regard to COX-1 and COX-2 ex vivo activities in the intestinal polyps. Administration of 20 p.p.m. p-XSC significantly inhibited COX-2 activity in the polyps in APCmin mice (P ⬍ 0.03), but it had no significant effect on COX-1 activity. Discussion
Fig. 2. Effect of p-XSC on colon tumor formation in APCmin mice. Detailed information is given in Materials and methods.
not produce any gross changes attributable to toxicity in liver, kidneys or lungs and also had no effect on body weight gain. Intestinal tumor incidence and multiplicity All APCmin mice developed intestinal tumors (100%) and most of these tumors (⬎95%) occurred in the small intestine. On average, these mice developed between 14 and 62 tumors/ mouse in the small intestine, whereas only between 0 and 6 tumors/mouse developed in the colon. All of the histopathologically classified tumors in the small intestine as well as those in the colon were adenomas (adenomatous polyps), with no evidence of local invasion of the lamina propria. Figures 1 and 2 summarize the chemopreventive effect of p-XSC with regard to formation of tumors in the small intestine and colon of APCmin mice. Dietary administration of 10 or 20 p.p.m. p-XSC significantly inhibited the multiplicity of adenomatous polyps in the small intestine (P ⬍ 0.003–0.0001). Also, mice fed pXSC had fewer colon tumors (mean 1.5/mouse in the 10 p.p.m. p-XSC group; 0.9/mouse in the 20 p.p.m. p-XSC group) when compared with the control diet group (2.8 tumors/ mouse). p-XSC produced dose-dependent tumor inhibition in both the small intestine (P ⬍ 0.0001) and the colon (P ⬍ 0.0035). Modulation of β-catenin and COX-2 activities To understand the mechanism of p-XSC-induced chemopreventive activity, we studied the expression and activity levels of β-catenin, COX-1 and COX-2 in intestinal polyps and in the intestinal mucosa of APCmin mice. Immunoblot analysis of β-catenin, COX-1 and COX-2 expression in small
This is the first study that demonstrates that p-XSC, a synthetic organoselenium compound, possesses chemopreventive activity against genetically predisposed neoplastic lesions associated with the APC gene in the intestine. This finding underscores the likelihood that p-XSC may be an effective chemopreventive agent in colorectal cancer prevention trials for patients with FAP. As part of a large scale investigation into the development of less toxic but highly effective organoselenium compounds as chemopreventive agents, we have shown previously that several organoselenium agents, including p-XSC, inhibit colon carcinogenesis in rats (15,21). Recently, we have shown that p-XSC administered along with a high fat diet significantly suppresses chemically induced colonic adenocarcinomas in male F344 rats. This effect was even more pronounced when p-XSC was combined with a low fat diet (18). In the present study, a low fat diet protocol might have provided greater efficacy than the high fat dietary regimen. However, in the current study a high fat dietary regimen was used to simulate the dietary fat consumption of Americans generally. It should be noted that the tumorinhibiting properties of p-XSC are not limited to colon cancer. In published studies, dietary supplementation with p-XSC during the initiation and/or post-initiation periods also significantly inhibited 7,12-dimethylbenz[a]anthraceneinduced mammary gland carcinogenesis in female rats (16). In another model assay system, lung tumor development by the tobacco-specific nitrosamine 4(methylnitrosamino)-1(3pyridyl)-1-butanone in A/J mice was inhibited by p-XSC, although sodium selenite and naturally occurring SM had no inhibiting effect (17). The results of this study and of earlier investigations with chemically induced cancers of the colon (18,21), mammary gland (16), lung (17) and oral cavity (20) testify to the efficacy of p-XSC in different types of chemically induced cancers in various species. Several epidemiological, experimental and clinical intervention studies support the important role of Se in cancer prevention (10,11,25,26). A comparison of p-XSC with other selenium compounds showed that the sub-chronic MTD dose of p-XSC in rats is 10-fold higher than that of Na2SeO3 and of benzyl selenocyanate and 4-fold higher than that of SM. Further, the preventive efficacy index (defined as the ratio of MTD to effective dose) of p-XSC is 6.5 in rats, compared 619
C.V.Rao et al.
Table I. Modulatory effects of p-XSC on β-catenin, COX-1 and COX-2 protein expression in intestinal mucosa and tumors of APCmin mouse β-Catenin
Group
Control 20 p.p.m. p-XSC aExpression levels bMean ⫾ SEM.
COX-1
COX-2
Mucosaa
Tumors
Mucosaa
Tumors
Mucosaa
Tumors
145 ⫾ 18b (6)d 131 ⫾ 16 (6)
274 ⫾ 24 (8) 128 ⫾ 14c (8)
22.6 ⫾ 4.7 (6) 23.8 ⫾ 5.1 (6)
58.3 ⫾ 7.5 (8) 54.7 ⫾ 8.2 (7)
14.2 ⫾ 3.4 (6) 12.6 ⫾ 3.5 (6)
42.1 ⫾ 6.2 (8) 36.9 ⫾ 7.1 (8)
(ng of β-catenin, COX-1 and COX-2 per mg protein) by western blot analysis as described in Materials and methods.
different from control diet group by Student’s t-test, P ⬍ 0.0001. in parentheses are numbers of samples analyzed per group.
cSignificantly dValues
Table II. Effects of p-XSC on the COX-1 and COX-2 activities in intestinal tumors of APCmin mouse Group
COX-1 activitya
COX-2 activitya
Control 20 p.p.m. p-XSC
22.8 ⫾ 4.0b (6)c 18.1 ⫾ 2.4d (6)
58.1 ⫾ 6.2 (6) 41.2 ⫾ 3.8ce (6)
aCOX-1, pmol [14C]PGs and TxB formed/mg protein/20 min; COX-2, 2 pmol [14C]15(R)-HETE formed/mg protein/20 min. The COX-1 and COX-2
specific assays are described in Materials and methods. bMean ⫾ SEM. dSignificantly different from control diet group by Student’s t-test, P ⫽ 0.16. cValues in parentheses are numbers of samples analyzed per group. eSignificantly different from control diet group by Student’s t-test, P ⬍ 0.03.
with indices of 1, 1.3 and 2.5 for SM, Na2SeO3 and benzyl selenocyanate, respectively. Importantly, the cancer preventive efficacy of p-XSC in different models (chemically induced as well as transgenic) and its low toxicity, in comparison with all previously known selenium compounds, provide a rational basis for designing chemoprevention strategies in the human setting with this agent. The precise mechanisms by which p-XSC inhibits the development of adenomatous polyps is not fully known, yet it is likely that this agent affects more than one stage of tumorigenesis. Interestingly, the findings in the present study suggest that p-XSC significantly suppresses membrane-bound (plasma and nuclear) β-catenin expression in intestinal polyps. It is well established that β-catenin plays an important role in cytoskeleton formation, cell adherence junctions and signal transduction by forming complexes with transcription factors (Tcf) in the nucleus (4,9). Importantly, disruption of APCmediated regulation of the β-catenin/Tcf/LEF-1 pathway is associated with development of colon tumors (4,7–9). Certain colorectal cancers that lack APC mutations may have functional mutations of β-catenin. Also, abnormal APC function is associated with elevated levels of intracellular β-catenin (27,28). As transcriptional activators, nuclear β-catenin–Tcf complexes may interact with DNA-binding proteins to modulate the expression of genes that regulate cellular events, such as apoptosis (4,28–30). Observations in the present study support the hypothesis that p-XSC-induced suppression of β-catenin expression may in part be responsible for its chemopreventive activity in APCmin mice. We have not assayed the effect of p-XSC on enterocyte apoptosis in the present study, however, studies in our laboratory have shown that this agent enhanced apoptosis in chemically induced colon tumors (31). Further studies are needed to understand the mechanism by which p-XSC suppresses β-catenin expression in colon tumors. 620
Several studies support the idea that overexpression of COX-2 is an early, central event in colon carcinogenesis (22,32). In the present study we have shown that p-XSC has no significant effect on COX-2 protein expression. However, we observed that administration of p-XSC caused a moderate decrease (P ⬍ 0.03) in COX-2 activity in polyps of APCmin mice. This observation suggests that p-XSC modulates COX-2 activity at the post-translational level rather than at the protein expression level. In addition to the possible modes of action mentioned above, p-XSC may act via general mechanisms of tumor inhibition that have been proposed for selenium compounds, including inhibition of lipid peroxidation and facilitation of peroxide decomposition, free radical scavenging, repair of molecular damage and incorporation into enzymes with protective functions for the cell, i.e. glutathione peroxidase (21,33), an enzyme responsible for preventing oxidative damage due to peroxidation. In conclusion, we have shown for the first time that dietary administration of p-XSC significantly suppresses the tumorigenic effects of a germline APC mutation in the Min mouse. The tumor inhibitory effects of p-XSC may also include modulation of β-catenin expression and COX-2 activity. While our understanding of the mechanisms of the chemopreventive action of p-XSC is evolving, the development of preventive strategies on the basis of the present and earlier experimental studies will serve as a practical approach towards the design of chemoprevention trials in humans. Acknowledgements The authors thank Joanne Braley and Jeff Rigotty of the Research Animal Facility and Beverly Gambrell of the Histopathology Facility for expert technical assistance. We thank Laura Nast for preparation of the manuscript and Ilse Hoffmann for editing it. This work was supported by USPHS grants CA-46589, CA 17613 and CA-80003 from the National Cancer Institute.
References 1. Landis,S.H., Murray,T., Bolden,S. and Wingo,P.A. (1999) Cancer Statistics, 1999. CA Cancer J. Clin., 49, 8–31. 2. Giovannucci,E. and Willett,W.C. (1994) Dietary factors and risk of colon cancer. Ann. Med., 26, 443–452. 3. Reddy,B.S. (1986) Diet and colon cancer: evidence from human and animal model studies. In Reddy,B.S. and Cohen,L.A. (eds) Diet, Nutrition and Cancer: A Critical Evaluation. Vol. I. Macronutrients and Cancer. CRC Press, Boca Raton, FL, pp. 47–65. 4. Kinzler,K.W. and Vogelstein,B. (1997) Cancer-susceptibility genes. Gatekeepers and caretakers. Nature, 386, 761–763 5. Moser,A.R., Pitot,H.C. and Dove,W.F. (1989) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science, 247, 322–324. 6. Su,L.-K., Kinzler,K.W., Vogelstein,B., Preisinger,A.C., Moser,A.R., Luongo,C., Gould,K.A. and Dove,W.F. (1992) Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science, 256, 668–670.
Prevention of polyps in APCmin mouse by p-XSC 7. Bilger,A., Shoemaker,A.R., Gould,K.A. and Dove,W.F. (1996) Manipulation of the mouse germline in the study of Min-induced neoplasia. Semin. Cancer Biol., 7, 249–260. 8. Oshima,M., Dinchuk,J.E., Kargman,S.L., Oshima,H., Hancock,B., Kwong,E., Trzaskos,J.M., Evens,J.F. and Taketo,M.M. (1996) Suppression of intestinal polyposis in Apc∆716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87, 803–809. 9. Korinek,V., Barker,N., Morin,P.J., Van Wichen,D.V., de Weger,R., Kinzler,K.W., Vogelstein,B. and Clever,H. (1997) Constitutive transcriptional activation by a β-catenin-Tcf complex in APC –/– colon carcinoma. Science, 275, 1784–1787. 10. Clark,L.C. (1986) The epidemiology of selenium and cancer. Fed. Proc., 44, 2584–2589. 11. El-Bayoumy,K. (1991) The role of selenium in cancer prevention. In DeVita,V.T.Jr, Hellman,S. and Rosenberg,S.A. (eds) Cancer Prevention. Lippincott, Philadelphia, PA, pp. 1–15. 12. Thompson,H.J., Meeker,L.D. and Kokoska,S. (1984) Effect of an inorganic and organic forms of dietary selenium on the promotional stage of mammary carcinogenesis in the rat. Cancer Res., 44, 2803–2806. 13. Fan,A.M. and Kizer,K.W. (1990) Selenium: nutritional toxicologic and clinical aspects. West. J. Med., 153, 160–167. 14. Ip,C. (1990) Activity of methylated forms of selenium in cancer prevention. Cancer Res., 50, 1206–1211. 15. Nayini,J.R., Sugie,S., El-Bayoumy,K., Rao,C.V., Rigotty,J., Sohn,O.-S., Fiala,E. and Reddy,B.S. (1991) Effect of dietary benzylselenocyanate on azoxymethane-induced colon carcinogenesis in male F344 rats. Nutr. Cancer, 15, 129–139. 16. El-Bayoumy,K., Chae,Y.-H., Upadhyaya,P., Meschtner,C., Cohen,L.A. and Reddy,B.S. (1992) Inhibition of 7,12-dimethylbenz[a]anthracene-induced tumors and DNA adduct formation in the mammary gland of female Sprague–Dawley rats by the synthetic organoselenium compound 1,4phenylenebis(methylene)selenocyanate. Cancer Res., 52, 2402–2407. 17. El-Bayoumy,K., Upadhyaya,P., Desai,D.H., Amin,S. and Hecht,S.S. (1993) Inhibition of 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone tumorigenicity in mouse lung by the synthetic organoselenium compound 1,4phenylenebis(methylene)selenocyanate. Carcinogenesis, 14, 1111–1113. 18. Reddy,B.S., Rivenson,A., El-Bayoumy,K., Upadhyaya,P., Pittman,B. and Rao,C.V. (1997) Chemoprevention of colon cancer by organoselenium compounds and impact of high- or low-fat diets. J. Natl Cancer Inst., 89, 506–512. 19. Reddy,B.S., Upadhyaya,P., Simi,B. and Rao,C.V. (1994) Evaluation of organoselenium compounds for potential chemopreventive properties in colon carcinogenesis. Anticancer Res., 14, 2509–2514. 20. Tanaka,T., Makita,H., Kawabata,K., Mori,H. and El-Bayoumy,K. (1997)
1,4-Phenylenebis(methylene) selenocyanate exerts exceptional chemopreventive activity in rat tongue carcinogenesis. Cancer Res., 57, 3644–3648. 21. Reddy,B.S., Rivenson,A., Kulkarni,N., Upadhyaya,P. and El-Bayoumy,K. (1992) Chemoprevention of colon carcinogenesis by the synthetic organoselenium compound 1,4-phenylenebis(methylene) selenocyanate. Cancer Res., 52, 5635–5640. 22. Rao,C.V., Rivenson,A., Simi,B., Zang,E., Kelloff,G., Steele,V.E. and Reddy,B.S. (1995) Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent. Cancer Res., 55, 1464–1472. 23. Lecomte,M., Laneuville,O., Ji,C., DeWitt,D.L. and Smith,W.L. (1994) Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. J. Biol. Chem., 269, 13207–13215. 24. Rao,C.V., Kawamori,T., Hamid,R. and Reddy,B.S. (1999) Chemoprevention of colonic aberrant crypt foci by an inducible nitric oxide synthase-selective inhibitor. Carcinogenesis, 20, 641–644. 25. Ganther,H.E. (1999) Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis, 20, 1657–1666. 26. Combs,G.F.Jr and Gray,W.P. (1998) Chemopreventive agents: selenium. Pharmocol. Ther., 79, 179–192. 27. Takahashi,M., Fukuda,K., Sugimura,T. and Wakabayashi,K. (1998) βCatenin is frequently mutated and demonstrates altered cellular location in azoxymethane-induced rat colon tumors. Cancer Res., 58, 42–46. 28. Munemitsu,A., Albert,I., Souza,B., Rubinfeld,B. and Polakis,P. (1995) Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc. Natl Acad. Sci. USA, 92, 3046–3050. 29. Wong,M.H., Hermiston,M.L., Syder,A.J. and Gordon,J.I. (1996) Forced expression of the tumor suppressor adenomatous polyposis coli protein induces disordered cell migration in the intestinal epithelium. Proc. Natl Acad. Sci. USA, 93, 9588–9593. 30. Morin.,P.J., Vogelstein,B. and Kinzler,K.W. (1996) Apoptosis and APC in colorectal tumorigenesis. Proc. Natl Acad. Sci. USA, 93, 7950–7954. 31. Samaha,H., Hamid,R., El-Bayoumy,K., Rao,C.V. and Reddy,B.S. (1997) The role of apoptosis in the modulation of colon carcinogenesis by dietary fat and by the organoselenium compound 1,4-phenylenebis(methylene) selenocyanate. Cancer Epidemiol. Biomarkers Prev., 6, 699–704. 32. Prescott,S.M. and White,R.L. (1996) Self-promotion? Intimate connections between APC and prostaglandin H synthase-2. Cell, 87, 783–786. 33. Rotruck,J.T., Pope,A.L., Ganther,H.E. and Hoekstra,W.G. (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science, 179, 588–590. Received July 7, 1999; revised October 13, 1999; accepted November 8, 1999
621
